JP2009523721A - Immunogenic substances comprising an adjuvant based on polyinosinic acid-polycytidylic acid - Google Patents

Abstract

  The present invention provides polynucleotide adjuvant (PICKCa) compositions and methods of use in eliciting an immune response, particularly a mucosal immune response. The polynucleotide adjuvant composition includes polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic, and at least one cation. The invention also includes polygenics together with other immunogenic compositions such as antigens selected from viral antigens, bacterial antigens, fungal antigens, parasite antigens and / or cancer antigens (such as those found in vaccines). An immunogenic composition comprising a nucleotide adjuvant composition is provided. The present invention further contemplates methods of using such adjuvant compositions, particularly in causing an immune response, particularly a mucosal immune response to an antigenic compound.

Description

Detailed Description of the Invention

(Field of the Invention)
The present invention generally relates to immunogenic compositions and methods using the same. More particularly, the invention relates to an immunogenic composition comprising a polynucleotide adjuvant combined with one or more antigenic substances used to elicit a disease specific immune response in a host.

BACKGROUND OF THE INVENTION
The immune system exhibits both specific and non-specific immunity. Nonspecific immunity includes, among other things, a variety of cells and mechanisms such as phagocytosis (uptake of foreign microparticles or antigens) by macrophages or granulocytes, and natural killer (NK) cell activity. Non-specific immunity relies on mechanisms that are not well evolved and does not exhibit the acquired nature of specificity and memory, typical features of a specific immune response. The main difference between specific and non-specific immunity is based on B cell and T cell specificity. These cells primarily have a mechanism that acquires their responsiveness after activation by specific antigens and presents memory when further exposed to this antibody. As a result, vaccination (including specificity and memory) is an effective protocol for protection against harmful pathogens.

  Normally, B lymphocytes and T lymphocytes present specific receptors for specific antigens on the cell surface, producing specific immunity. The specific immune system can respond to different antigens by two means: 1) humoral immunity, including B cell stimulation and production of antibodies or immunoglobulins by B cells, antigens and helper T cells (mainly Th2), And 2) Cellular immunity, which typically includes T cells, including cytotoxic T lymphocytes (CTLs), and also includes other cells involved in the production of CTL responses (eg, antigen presenting cells and Th1 cells).

  In ongoing research seeking safer and more effective vaccines, new technologies, including recombination, purification and synthesis methods, are used to improve quality and specificity for the antigens used. Purified, subunitized and synthetic antigens showed improved safety, but reduced immunogenicity, one driver for identifying effective adjuvants. Effective adjuvants are therefore increasingly an essential component of modern vaccines. An adjuvant is generally a composition that enhances and / or modifies the immune response to a particular antigen when administered with the antigen (simultaneously with or at a predetermined time prior to administration of the antigen). It is.

  Typical adjuvants used to enhance the immune response include aluminum compositions (all commonly referred to as “alum”), oil-in-water emulsions (Freund Complete Adjuvant (CFA) is an oil-in-water emulsion containing Mycobacterium tuberculosis organism that has been dried and heat-killed, saponin (Quillaja Saponoria) isolated from the bark of Quile A Monophosphoryl lipid A (MPL) derived from lipopolysaccharide of Salmonella minnesota Re595, CpG ODN (a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotide), ), Liposomes (usually by biodegradable substances such as phospholipids) Ri has been issued), and a biodegradable polymer microspheres (made by various polymers, such as polyphosphazene and polyanhydrides). The adjuvant properties of these compounds have been evaluated as each adjuvant exhibits advantages and disadvantages.

  Polynucleotide conjugates have been investigated for a variety of applications, including acting as adjuvants. Double-stranded RNA (dsRNAs) are very powerful biological modifiers that can have a profound effect on cells at nanomolar concentrations. Modulatory effects by dsRNA span a wide range of actions at the molecular and cellular levels.

  At the molecular level, dsRNAs can cause biological effects such as interferon synthesis, induction of protein kinases, enhancement of histocompatibility antigens and metabolic inhibition. Also, at the cellular level, dsRNA is an organism such as pyrogenicity, mitogenicity, macrophage activation, humoral immunity activation, cellular immunity activation and antiviral state induction. Can cause pharmacological effects. The immunomodulatory effect of dsRNAs has already been disclosed. US Pat. No. 4,124,702 discloses that double-stranded polynucleotides induce interferon induction in living animal cells. US Pat. No. 3,906,092 discloses that the antibody response to an adjuvanted vaccine is increased by binding to a polynucleotide or polynucleotide conjugate vaccine. Houston et al. Constructed PICLC (polyinosinic acid polycytidylic acid poly-L-lysinecarboxy-methylcellulose complex) as a potent adjuvant by increasing the primary antibody response without adding new adjuvants.

  Polyinosinic acid-polycytidylic acid (PIC) is one of the most studied polynucleotide complexes, but when used in monkeys and humans, it is effective because it is unstable in the body after administration. It wasn't. Therefore, PIC modifications have been made by various techniques to overcome one or other deficiencies. For example, a complex of polyriboinosinic acid-polyribocytidylic acid and poly-L-lysine hydrobromide is approximately 5 to 15 times more hydrolyzed by pancreatic ribonuclease compared to the original PIC. Resistant.

  Lin et al. Report that antiviral drugs including polyinosine polycytidylic acid, kanamycin and calcium can be used as adjuvants (Lin, et al., A new immunostimulatory complex (PICKCa) in experimental rabies: antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993; and Chinese Patent No. 93105862.7). Chinese Patent No. 93105862.7 provides the use of a general composition of poly I: C, kanamycin and calcium (PICKCa) as an adjuvant in vaccines for human and mammalian use. However, the originally identified form of PICKCa does not provide optimal efficiency / safety features for use of adjuvants, and it also contains unacceptable side effects under certain circumstances. Lin found out.

  The present invention provides novel immunogenic compositions with improved safety and efficiency features; and methods of using the compositions. The main immunogenic composition includes a polynucleotide adjuvant and an antigen.

[Reference]
The following references are noted:
・ Japanese Unexamined Patent Publication No. 01-93540 ・ US Pat. No. 4,124,702 ・ US Pat. No. 3,692,899 ・ US Pat. No. 3,906,092 ・ US Pat. No. 4,389,395 US Pat. No. 4,349,538, US Pat. No. 4,024,241, US Pat. No. 3,952,097, Houston et al., Infection and Immunity, 14: 318-9, 1976C
・ Wright and Adler-Moore, Biochemical and Biophysical Research Communications, 131: 949-45, 1985
Lin, et al., A new immunostimulatory complex (PICKCa) in experimental rabies; antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993
・ Chinese Patent No. 93105862.7 ・ Gupta RK et al., Adjuvants-a balance between toxicity and adjuvanticity, Vaccine, 11: 293-306, 1993
Arnon, R. (Ed.) Synthetic Vaccines 1: 83-92, CRC Press, Inc., Boca Raton, Fla., 1987
・ Sela, M., Science 166: 1365-1374 (1969)
・ US Patent No. 6,008,200 ・ Ellouz et al., Biochem. & Biophy. Res. Comm., 59: 1317, 1974
・ US Pat. No. 4,094,971 ・ US Pat. No. 4,101,536 ・ US Pat. No. 4,153,684 ・ US Pat. No. 4,235,771 ・ US Pat. No. 4,323,559・ US Pat. No. 4,327,085 ・ US Pat. No. 4,185,089 ・ US Pat. No. 4,082,736 ・ US Pat. No. 4,369,178 ・ US Pat. No. 4,314,998・ US Pat. No. 4,082,735 ・ US Pat. No. 4,186,194 ・ US Pat. No. 6,468,558 ・ New Trends and Development in Vaccines, edited by Voller et al., University Park Press, Baltimore , Md., USA, 1978
・ Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston (1997)
・ Gupa RK and Siber GR, Adjuvants for human vaccines-current status, problems and future prospects, Vaccine, 13 (14): 1263-1276, 1995
· Richard T Kenney et al Meeting Report -. 2 nd meeting on novel adjuvants currently in / close to human clinical testing, Vaccine 20 2155-2163, 2002
・ Laboratory Techniques in Rabies Edited by FX Meslin, MM Kaplan, H Koprowski 4 ^th , 1996, Edition ISBN 92 4 1544 1

[Summary of the Invention]
In general, the present invention relates to novel immunogenic compositions comprising a polynucleotide adjuvant together with an immunogenic or antigenic substance, and methods of use that elicit an immune response.

  Thus, an immunogenicity comprising (a) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation, and (b) at least one antigen. An immunogenic composition is provided, wherein the composition is formulated for sustained release administration.

  The immunogenic composition according to the present invention comprises polynucleotide adjuvant composition molecules having a non-uniform molecular weight, the molecular weight being at least 66,000 daltons.

[Brief description of the drawings]
FIG. 1-ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with vaccines containing PIKA and / or HBs antigen adw type.
FIG. 2-ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with vaccines containing PIKA and / or HBs antigen adw type.
FIG. 3-ELISPOT detection of IL-4 produced by mouse splenocytes after immunization with vaccines containing PIKA and / or HBs antigen adw type.
FIG. 4-ELISA detection of specific IgG titers from mouse serum (400x dilution) after immunization with vaccines containing PIKA and / or HBs antigen adw type.
FIG. 5-ELISPOT detection of mouse splenocytes that produced interferon-gamma (γ) after immunization with vaccines containing PIKA and / or inactivated and split influenza antigen.
FIG. 6-ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with vaccines containing PIKA and / or inactivated and split influenza antigens.
FIG. 7—ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with vaccines containing PIKA and / or inactivated and split influenza antigen.
FIG. 8—ELISA detection of specific IgG titers derived from mouse sera (900 × dilution) after immunization with vaccines containing PIKA and / or inactivated and split influenza antigen.
FIG. 9—ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with vaccines containing PIKA and / or HIV gp120 antigen.
FIG. 10—ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with vaccines containing PIKA and / or HIV gp120 antigen.
FIG. 11—ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with vaccines containing PIKA and / or HIV gp120 antigen.
FIG. 12-FACS analysis in mouse splenocytes after immunization with vaccines containing PIKA and / or HIV gp120 antigen and percentage of CD4 + cells expressing interferon-γ.
FIG. 13—ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with vaccines containing PIKA and / or anthrax rPA antigen.
FIG. 14—ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with vaccines containing PIKA and / or anthrax rPA antigen.
FIG. 15—ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with vaccines containing PIKA and / or anthrax rPA antigen.
FIG. 16-FACS analysis in mouse splenocytes after immunization with vaccines containing PIKA and / or anthrax rPA antigen and percentage of CD4 + cells expressing interferon-γ.
FIG. 17—ELISA detection of specific IgG titers from mouse sera (400 × dilution) after immunization with vaccines containing PIKA and / or anthrax rPA antigen.
FIG. 18-ELISA detection of specific IgG titers from mouse serum 16 weeks after immunization with vaccines containing PIKA and / or anthrax rPA antigen.
FIG. 19—ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with vaccines containing PIKA and / or HSV2-gD antigen.
FIG. 20—ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with vaccines containing PIKA and / or HSV2-gD antigen.
FIG. 21—ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with vaccines containing PIKA and / or HSV2-gD antigen.
FIG. 22-FACS analysis in mouse splenocytes after immunization with vaccines containing PIKA and / or HSV 2gD antigen, percentage of CD4 + cells expressing interferon-γ.
FIG. 23—ELISA detection of specific IgG titers derived from mouse serum (2,700 × dilution) after immunization with vaccines containing PIKA and / or HSV2-gD antigen.
FIG. 24-ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen.
FIG. 25—ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen.
FIG. 26—ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen.
FIG. 27-FACS analysis in mouse splenocytes after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen, percentage of CD4 + ve cells expressing interferon-γ.
FIG. 28-ELISA detection of specific IgG titers from mouse serum (900 × dilution) after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen.
FIG. 29-ELISA detection of specific IgG titers derived from mouse sera (16,000 dilution) after immunization with vaccine containing whole PIKA and / or inactivated SARS antigen.
FIG. 30-ELISA detection of specific antibody H5 titers from chicken serum after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen.
FIG. 31-ELISA detection of specific H9 antibody titers from chicken serum after immunization with vaccines containing PIKA and / or inactivated H9N2 antigen.
FIG. 32-Survival rate of mice prescribed rabies vaccine after exposure to wild rabies virus.
FIG. 33-ELISA detection of specific antibody titers from mouse sera after immunization with vaccines containing PIKA and / or HBs antigen adw.
FIG. 34-ELISA detection of specific antibody titers from mouse sera after immunization with vaccines containing PIKA and / or inactivated and split influenza antigen.
FIG. 35-ELISA detection of specific IgG1 titers from mouse sera after immunization with vaccines containing PIKA and / or HBs antigen.
FIG. 36—ELISA detection of specific IgG2 titers from mouse sera after immunization with vaccines containing PIKA and / or HBs antigen.
FIG. 37—ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with vaccines containing PIKA and / or HBs antigen.
FIG. 38-ELISPOT detection of mouse splenocytes (after 6 days restimulation with 2 ug / ml IPQ peptide) after immunization with vaccines containing PIKA and / or HBs antigen.
Table 1 is a table of typical viral pathogens and diseases associated with these organisms that may serve as a source of antigen.
Table 2 is a table of typical bacterial pathogens that can serve as antigen sources and diseases associated with these organisms.
Table 3 is a table of typical fungal pathogens that can serve as antigen sources and diseases associated with these organisms.
Table 4 is a table of typical parasites that can serve as antigen sources and the diseases associated with these organisms.
Table 5 is a table of typical cancers (eg, by tissue) that may serve as antigen sources.

Detailed Description of Embodiments Illustrating the Invention
The present invention will be understood more readily by reference to the following detailed description of specific embodiments of the invention and the examples described herein.

  This application lists references, and the disclosures of these documents are incorporated herein by reference in their entirety to better describe the state of the art to which the present invention pertains.

  Before further describing the present invention, it is to be understood that the present invention is not limited to the specific embodiments used in the description, and may, of course, vary. Since the scope of the present invention is limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Should also be understood.

  Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or evaluation of the present invention, the preferred methods and materials are now described. All references cited herein are hereby incorporated by reference herein in conjunction with the methods and / or materials listed therein to disclose and explain the methods and / or materials. Is done.

  As used herein in the specification and in the claims, the singular forms “a”, “and” and “that” unless the context clearly indicates otherwise. It should be noted that it includes plural objects. Thus, for example, reference to “a single immunogenic composition” includes a plurality of such compositions, and “an antigen” refers to one or more antigens and is known to those skilled in the art. And other equivalents. It should further be noted that the claims may be drafted without considering any component. As such, this specification uses the exclusive terms such as “one” and “only”, or a “passive” limitation, with an enumeration of the components of the claim. It is intended to serve as a basis for.

<Definition of terms>
Before describing the details of the present invention, it may be useful to understand the present invention to provide definitions for some terms used herein.

As used herein, the term “adjuvant” refers to any substance or mixture of substances that increases or diversifies a host's immune response to an antigenic compound.
In particular:
1. The term “PICKCa” generally refers to a composition comprising poly I: C, kanamycin and calcium, regardless of specific physical and immunogenic properties.
2. “Av-PICKCa” refers to a form of PICKCa that is commercially used as an antiviral agent.
3. “PIKA” refers to a composition of the invention comprising poly I: C, antibiotics (eg, kanamycin), cations (eg, calcium), PIKA is a substance property (eg, molecular weight, size, etc.) as administered. ), And PIKA has less side effects than PICKCa for example (eg reduced toxicity) and is more effective than eg Av-PICKCa (eg enhanced immunity) (Stimulates the response).

  The term “poly I: C” or “PIC” refers to a composition comprising a polyriboinosine nucleic acid and a polyribocytidyl nucleic acid (also referred to as polyinosinic acid-polycytidylic acid, respectively).

  “PIC-containing molecule” or “PIC-containing compound” refers to PIC without limitation, and includes at least one of antibiotics (eg, kanamycin) and cations (eg, calcium) present in a composition comprising a PIC-containing molecule. Combined with one or both, depending on the situation, or otherwise combined. In one embodiment, the PIC-containing molecule does not include poly-L-lysine or a derivative thereof in the complex.

  As used herein, “heterogeneous” as used in the context of the adjuvant composition of the present invention means that the constituents of the composition, for example PIC-containing molecules, are homogeneous with respect to the physical properties of molecular weight, size or both. It means not. When a composition is described as being heterogeneous in a given physical property, and further when the composition is described as being a range of values in its physical property, it covers the listed ranges. And is believed to consist essentially of molecules characterized by molecules having physical properties that are beyond range. Although the composition does not include molecules corresponding to all physical property values between the upper and lower limits of the recited range, the composition generally comprises molecules having upper and lower physical properties. Will contain at least one. The composition in certain embodiments may include molecules that are outside the defined range of physical properties used to describe the composition. Molecules outside the defined range in the composition do not substantially affect the basic and novel properties of the composition.

  The term “individual” is used herein interchangeably with “host”, “subject” and “animal”, and includes humans and all domesticated and wild mammals such as domestic animals and pets. , As well as poultry, including but not limited to cattle, horses, dairy cows, pigs, sheep, goats, dogs, cats, rabbits, deer, mink, chickens, ducks, geese, turkeys and gamehens It is not a thing.

  The term “antibody” includes polyclonal and monoclonal antibodies, as well as antigenic substance binding fragments of antibodies such as those comprising Fab, F (ab ′) 2, Fd and Fv fragments, and single chains thereof. Contains derivatives. Furthermore, the term “antibody” includes naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies and related synthetic isoforms. The term “antibody” is used interchangeably with “immunoglobulin”.

  As used herein, an “antigenic compound” is any substance that can be recognized by the immune system under appropriate conditions (eg, processed to bind an antibody or cause an intracellular immune response). Point to.

  As used herein, an “antigen” is a cell, cell extract, protein, lipoprotein, glycoprotein, nucleoprotein, polypeptide, peptide, polysaccharide, polysaccharide complex, polysaccharide mimic by peptide, lipid, sugar Including, but not limited to, lipids, carbohydrates, viruses, virus extracts, bacteria, bacterial extracts, fungi, fungal extracts, parasites and other multicellular organisms, and allergens. The antigen can be foreign (eg, from something other than the individual to whom the antigen is administered, eg, from a different species), or endogenous (eg, from within the host, eg, a diseased component of the body, Cancer antigens, and virus-infected cells that produce the antigens). Antigens can be natural (eg, naturally occurring), synthetic, or recombinant. Antigens include crude extracts, whole cells, and purified antigens. As used herein, “purified” refers to an antigen in a state where the antigen normally occurs and / or in a state that is concentrated compared to a crude extract such as, for example, a culture state of the antigen.

  As used herein, an “immunogenic composition” refers to a combination of two or more substances (eg, an antigen and an adjuvant) that together cause an immune response when administered to a host.

  The terms “polypeptide”, “peptide”, “oligopeptide” and “protein” are used interchangeably herein to refer to a polymeric form of any length of amino acids, encoding amino acids and encodings. Unmodified amino acids, chemically modified or biochemically modified amino acids or derived amino acids, and polypeptides having modified peptide backbones.

  “An effective amount of an antigenic compound” refers to the amount of an antigenic compound that, when combined with an adjuvant, will cause a subject to generate a specific immune response against the antigenic compound.

The term “immune response” refers to any response to an antigenic or immunogenic compound by the immune system of a vertebrate subject. Typical immune responses include CTL responses including antigen-specific induction of CD8 ⁺ cytotoxic T lymphocytes (CTLs), helper T cell responses including T cell proliferation and cytokine release, and B cell responses including antibody responses. Local and systemic cellular immunity and humoral immunity, such as, but not limited to.

  The term “inducing an immune response” is used herein primarily to encompass the induction and / or enhancement effects of an immune response.

  The term “inducing an immune response” refers to a stimulated, triggered or induced immune response.

  The term “enhancing an immune response” refers to an existing immune response that has been improved, promoted, supplemented, amplified, enhanced, augmented, or prolonged.

  The expression “enhanced immune response” or similar expression increases the benefits of an immune response to the host compared to a state of a prior immune response, eg, prior to administration of an immunogenic composition of the invention, Improves and means enhancement.

  The terms “humoral immunity” and “humoral immune response” refer to the form of immunity in which antibody molecules are produced in response to antigenic stimulation.

  The terms “cellular immunity” and “cellular immune response” refer to immune protection provided by lymphocytes, such as, for example, immune protection provided by T cell lymphocytes when T cell lymphocytes are in close proximity to the victim cell. Means. A cellular immune response usually involves the proliferation of lymphocytes. When “lymphocyte proliferation” is measured, the proliferative capacity of lymphocytes in response to a particular antigen is measured. Lymphocyte proliferation is meant to refer to B cell, T helper cell, or cytotoxic T lymphocyte (CTL) cell proliferation.

  The term “immunogenic amount” refers to an antigen that is sufficient to stimulate an immune response when administered a primary immunogenic composition as compared to an immune response elicited by the antigen in the absence of a polynucleotide adjuvant. Refers to the amount of the active compound.

  The term “immune enhancing amount” refers to an antibody as compared to an increase in antibody and / or cellular immunity levels observed in the absence of a polynucleotide adjuvant when an antigenic compound in the composition of the invention is administered. Refers to the amount of adjuvant required to bring about an increase in titer and / or cellular immunity.

  The terms “treatment”, “treating”, “treating” and the like are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. This effect may be a prophylactic effect in terms of completely or partially suppressing the disease or disease symptoms and / or partially or fully stabilizing the disease and / or side effects resulting from the disease It may be a therapeutic effect from the viewpoint of making or healing. As used herein, “treatment” includes any treatment of a subject, particularly a mammalian subject, and more particularly a human disease, and includes: (a) suffering from a disease or condition Reduce the occurrence of a disease or symptom in a subject who has not yet been diagnosed as having an easy, but (b) prevent the symptom of the disease, eg, prevent its development, or reduce the symptom of the disease Preferably, to cause regression of the disease or condition, (c) to reduce the level of products (eg, toxins and antigens) produced by the pathogen causing the disease, and (d) to the pathogen causing the disease To reduce no physiological response (eg fever and tissue edema).

  As used herein, the term “mixing” is meant to include any method of combining the components of the composition. Such methods include, but are not limited to, mixing, distributing, dissolving, emulsifying, coagulating or suspending the components of the composition, or another method of physically mixing the components of the composition. It is not something.

  A “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and retains the desired pharmacological activity of the underlying compound. Such salts include the following: (1) a salt to which an acid has been added, formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, or Acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxy Benzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4′-methylenebis- (3-hydroxy 2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid. Or (2) replacing acidic protons in the original compound with metal ions such as alkali metal ions, alkaline earth ions or aluminum ions, or ethanolamine, diethanolamine, triethanolamine , A salt formed by either using an organic base such as tromethamine and N-methylglucamine.

  As used herein, the term “unit dosage form” refers to a physically discrete unit suitable as a single dosage for human and animal subjects, wherein each unit is a pharmaceutical / With a physiologically acceptable diluent, carrier or excipient, it contains a defined amount of a compound of the present invention, calculated in an amount sufficient to produce the desired effect.

<Exemplary Embodiment of the Present Invention>
The present invention is directed to immunogenic compositions and methods useful for inducing and / or enhancing immune responses, which are humoral and / or cellular in humans, non-human animals or cell cultures. It may be an immune response. In general, the primary immunogenic composition comprises an antigen ("antigenic composition") and an adjuvant. The presence of an adjuvant enhances and modifies the immune response to the antigen. Adjuvants may alter the quality of the immune response by affecting the subclasses (isotypes) of the immunoglobulins, chemokines and / or cytokines that are produced. As a result, innate, humoral and / or cellular immune responses are more effective due to the presence of adjuvants.

  A striking advantage is the effect of PIKA adjuvants in combination with antigenic substances in inducing specific humoral immune responses and thereby enhancing protective immunity.

  A further important advantage is the specific cellular immune response that PIKA adjuvants in combination with antigens are essential for therapeutic vaccines to limit and treat intracellular infections of viruses, bacteria and parasites, and cancer or It is possible to induce a specific cellular immune response essential for the treatment of diseases such as the treatment of autoimmune diseases.

  Accordingly, the present invention addresses the need for a safe adjuvant that has unique product characteristics that make it most suitable for use as a vaccine for administration to animals and / or humans and that elicits a beneficial immune response. Corresponding compositions are included.

  Accordingly, the present invention provides adjuvants and immunogenic compositions that can be safely utilized in humans and animals.

  Thus, an immunogenicity comprising (a) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation, and (b) at least one antigen. An immunogenic composition is provided, wherein the composition is formulated for sustained release administration.

  The immunogenic composition according to the present invention may comprise polynucleotide adjuvant composition molecules having a non-uniform molecular weight, the molecular weight being at least 66,000 daltons. A value of 66,000 daltons corresponds to a size of about 6.4 Svedberg. Thus, a molecular weight in the range of 66,000 to 1,200,000 daltons corresponds to a size of about 6.4 to 24.0 Svedberg.

  In some embodiments, the PIKA adjuvant composition comprises a polynucleotide, an antibiotic and a cation, wherein the polynucleotide may be polyriboinosinic acid-polyribocytidylic acid (PIC) and the antibiotic is kanamycin. There may be, and the ion may be calcium.

  In one aspect of particular interest, the present invention provides an immunogenic composition for enhancing the antigenicity of an antigenic compound, comprising a polynucleotide adjuvant composition capable of causing an antigen-specific cellular immune response To do.

  In one aspect of particular interest, the present invention provides an immunogenic composition for enhancing the antigenicity of an antigenic compound, comprising a polynucleotide adjuvant composition capable of causing an antigen-specific humoral immune response To do.

  In one aspect of particular interest, the present invention enhances the antigenicity of an antigenic compound comprising a polynucleotide adjuvant composition capable of causing a combined response of a specific cellular immune response and a humoral immune response An immunogenic composition is provided.

  In one aspect of particular interest, the present invention provides a lyophilized adjuvant composition or a lyophilized immunogenic composition comprising the adjuvant composition.

  In one aspect of particular interest, the present invention provides the use of a polynucleotide adjuvant composition to prepare a medicament for enhancing an immune response in a host.

<Polynucleotide Adjuvant>
A subject immunogenic composition comprises a PIC-containing polynucleotide adjuvant. For example, PIKA compositions are generally comprised of polyinosinic acid, polycytidylic acid, antibiotics (eg, kanamycin), and divalent cations (eg, calcium). As a typical example of such PIC-containing adjuvants, reference is made herein to PIKA.

  The noted PIC-containing adjuvant can be produced using a technically available method. The PIC-containing adjuvant composition can be produced by any suitable process. For example, a polynucleotide adjuvant composition can be prepared by mixing polyinosinic acid, posocitidylic acid, antibiotics, and a cation source in a sodium chloride / phosphate buffer at pH 6-8. Polyinosinic acid and polycytidylic acid are generally provided at a concentration of 0.1 to 10 mg / ml, usually 0.5 to 5 mg / ml, more usually 0.5 to 2.5 mg / ml. The hyperchromicity value should be greater than 10%, greater than 15%, greater than 20%, or greater than 50%. The preparation of PIC and the combination of antibiotics (eg, kanamycin) and cations (eg, calcium) are generally performed under quality standards consistent with the Good Manufacturing Process.

  In certain embodiments of the invention, the antibiotic component of the adjuvant is kanamycin. In some embodiments where the antibiotic is kanamycin, the kanamycin in the polynucleotide adjuvant composition is used with or substituted with one or more antibiotics. One or more antibiotics include tobramycin), anthracycline, butirosin sulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrzamide, neomycin, puromycin, streptomycin and streptozocin Selected from the group. Antibiotics (eg, kanamycin or the like) in the polynucleotide adjuvant compositions of the invention will generally be about 10-100,000 units / ml, about 100-10,000 units / ml, or about 500-5,000 units. / Ml concentration.

  In certain embodiments of the invention, the polynucleotide adjuvant composition further comprises a cation (cation), usually a divalent cation, usually an alkali metal cation. Cations are generally provided in the compositions of the present invention as a cation source, such as a salt or complex. Examples of the salt or complex are an organic salt or an inorganic salt, or an organic complex or an inorganic complex, and usually an inorganic salt or an inorganic complex. Typical cations include, but are not necessarily limited to calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc.

  The cation can be provided in the form of any suitable salt or organic complex. Any suitable salt or organic complex includes, but is not necessarily limited to, chloride, fluoride, hydroxide, phosphate, or sulfate. For example, if the cation is calcium, the ions can be supplied in the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate, or calcium sulfate.

  Cations (e.g., calcium) are present at concentrations of about 10 umol / ml to 10 mmol / ml, usually about 50 umol / ml to 5 mmol / ml, more usually about 100 umol / ml to 1 mmol / ml. It can be supplied in the composition. The term “umol” is used in the text to denote micromolar.

  When the cation in the adjuvant composition of the present invention is calcium, the calcium ion can be combined with other cations or substituted with other cations. Other cations include cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron and zinc, and these ions exist in the form of inorganic salts or organic complexes. be able to.

  The resulting composition is a PIC-containing adjuvant further comprising an antibiotic and a cation. In some embodiments, the antibiotic is kanamycin and the ion is calcium, the product may be described as PICKCa. In related embodiments, the PICKCa composition can include molecules without limitation of different physical properties.

<PIKA adjuvant composition>
In certain exemplary embodiments, the PIC-containing adjuvant is PIKA. PIKA may be produced in various ways at the same time as it is produced from PICKCa of particular interest. PIKA can be produced from PICKCa through additional manufacturing steps that include isolation and / or enrichment of molecules of well-defined molecular size and / or molecular weight. Separation and concentration of polynucleotide molecules of a particular nature are performed using filtration, chromatography, heat treatment, centrifugation, electrophoresis, and similar methods using standard steps known to those skilled in the art.

  In a particular embodiment of interest, the invention features an adjuvant commonly referred to as PIKA. PIKA includes polyriboinosinic acid-polyribocytidylic acid (PIC), antibiotics (eg, kanamycin), and positively charged ions (eg, calcium ions), and the composition is about 66,000-1 , 200,000 daltons of heterogeneous molecular weight adjuvant molecules. That is, the adjuvant composition includes molecules having a weight distribution in the range of about 66,000 to 1,200,000 daltons.

  In related embodiments, the PIKA polynucleotide adjuvant composition molecules in the composition are heterogeneous. That is, the weight of the adjuvant molecule is about 300,000 to 1,200,000 daltons, or about 66,000 to 660,000 daltons, or about 300,000 to 660,000 daltons, or about 300,000 to 2, 000,000 daltons, or about 66,000-100,000 daltons, or 100,000-200,000 daltons, about 300,000-4,000,000 daltons, or about 500,000-1,000,000 daltons Or about 1,000,000 to 1,500,000 daltons, or about 1,500,000 to 2,000,000 daltons, or about 2,000,000 to 2,500,000 daltons, or about 2, 500,000-3,000,000 daltons, or about 3,000,00 ~ 3,500,000 daltons, or about 3,500,000-4,000,000 daltons, or about 4,000,000-4,500,000 daltons, or about 4,500,000-5,000, It is distributed in the range of 000 daltons.

  In related embodiments, the PIKA polynucleotide adjuvant composition molecule in the composition has an average molecular weight equal to, equal to or greater than 66,000 daltons, an average molecular weight greater than 150,000 daltons, or equal to 250,000 daltons Or an average molecular weight equal to or greater than, or an average molecular weight equal to or greater than 350,000 daltons, or an average molecular weight equal to or greater than 500,000 daltons, or an average molecular weight equal to or greater than 650,000 daltons, or An average molecular weight equal to or greater than 750,000 Daltons, or an average molecular weight equal to or greater than 1,000,000 Daltons, or equal to 1,200,000 Daltons Ku has a larger average molecular weight, or equal to 1,500,000 daltons or an average molecular weight greater than, or 2,000,000 Daltons equal or an average molecular weight greater than.

  In an embodiment of particular interest, the invention features an adjuvant commonly referred to as PIKA. PIKA includes polyriboinosinic acid-polyribocytidylic acid (PIC), antibiotics (eg, kanamycin), and positively charged ions (eg, calcium ions), and the composition contains a heterogeneous adjuvant molecule. Including. That is, the size of the adjuvant molecules is distributed over a range of molecular sizes having a sedimentation coefficient Svedbergs (S) of about 6.43S to 24.03S.

  In related embodiments, the PIKA polynucleotide adjuvant composition molecules in the composition are heterogeneous. That is, the size of the adjuvant molecule is from about 12.8S to 24.03S, or from about 3S to 12S, or from about 6.43S to 18.31S, or from about 12.8S to 18.31S, or from about 12.8S. 30.31S, or about 12.8S to 41.54S, or about 13.5S to 18.31S, or about 13.5S to 24.03S, or about 16.14S to 22.12S, or about 22.12S 26.6S, or about 26.6S-30.31S, or about 30.31S-33.55S, or about 33.55S-36.45S, or about 36.45S-39.1S, or about 39.1S- It is distributed in the range of 41.54S, or about 41.54S to 43.83S, or about 43.83S to 45.95S.

  In further related embodiments, the PIKA polynucleotide adjuvant composition has an average sedimentation coefficient (Svedberg) greater than 9, or an average sedimentation coefficient greater than 12, or an average sedimentation coefficient greater than 13.5, or an average sedimentation coefficient greater than 15. Or an average sedimentation coefficient greater than 16, or an average sedimentation coefficient greater than 17, or an average sedimentation coefficient greater than 18, or an average sedimentation coefficient greater than 19, or an average sedimentation coefficient greater than 20, or an average sedimentation coefficient greater than 21, or It has an average sedimentation coefficient greater than 22, or an average sedimentation coefficient greater than 25, or an average sedimentation coefficient greater than 30.

<Immunogenic properties>
An immunogenic composition comprising PIKA and an antigen can generally induce an antigen-specific immune response in at least two ways. That is, i) humoral immunity and ii) cellular immunity. Humoral immunity includes B cell stimulation and production of antibodies or immunoglobulins (other cells are also involved in the generation of antibody responses, eg, antigen presenting cells including macrophages and helper T cells (Th1 and Th2)). Cellular immunity generally involves T cells, including cytotoxic T lymphocytes. However, other cells are also involved in the generation of cytotoxic T lymphocyte responses (eg, Th1 and / or Th2 cells, and antigen presenting cells).
Furthermore, polynucleotide adjuvant compositions can alter the quality of the immune response by affecting the subclass (isotype) of the immunoglobulin produced, as well as the affinity of the immunoglobulin.

  Therefore, the extent and nature of the immunoglobulin response induced by the subject immunogenic composition can be assessed by measuring the presence of molecules including cytokines, chemokines, and antibodies produced by cells of the immune system. it can.

  Interleukin-4 is mainly produced by activated Th2 cells. The production of interleukin-4 (IL-4) induces B cell activation and, as a result, induces the production of IgG1 and IgE immunoglobulins (antibodies). Immunoglobulins can be measured in serum samples. IL-4 is considered an indicator of the Th2 immune response and a typical cytokine. Th2 cells tend to promote antibody responses.

  Interleukin-2 (IL-2) is produced primarily by activated Th1 cells, as well as NK cells and lymphokine activated killer (LAK) cells. IL-2 is involved in T cell proliferation and maturation, an essential step in an effective cellular adaptive immune response.

Interferon-γ (INF-γ) is produced by a variety of cells, including natural killer cells, as well as CD4 ⁺ and CD8 ⁺ T cells, and is an adaptive immune response that includes activating macrophages and making them highly bactericidal. Is an essential part. In addition, INF-γ is a factor that acts to specifically direct the development of Th1 T cells and consequently up-regulate the cellular adaptive immune response.

  The present invention contemplates methods of using the polynucleotide adjuvants of the present invention and antigens, for example, to elicit an antigen-specific humoral response and / or a specific cellular response (eg, T cells) in a subject. is doing. The immune response elicited may be a response to an antigen in an unimmunized subject or may play a role in enhancing the current immune response (eg, by booster immunization). An immunogenic composition comprising PIKA according to the present invention has particularly advantageous properties as described herein.

  A variety of different antigens were tested in vivo for the ability to induce an immune response, with or without PIKA. Test antigens include hepatitis B surface antigen adw type recombinant protein, inactivated split influenza vaccine (VAXIGRIP manufactured by Sanofi Pasteur), synthetic HIV peptide antigen, recombinant protein of herpes simplex virus type 2 gD antigen, recombinant protective anthrax protein antigen Avian influenza antigen H5N1 strain inactivated whole virus and antigen inactivated severe acute respiratory syndrome (SARS) inactivated whole virus.

  In each case, the expression of cytokines was enhanced by the presence of the PIKA adjuvant along with the antigen as compared to the case of the antigen alone or PIKA alone. In particular, increased expression of cytokines INF-γ, IL-2, and IL-4 (see Examples 1.1, 1.2, 1.3, 1.4, 1.5, and 1.6) The presence of the PIKA adjuvant suggests that the stimulation of specific adaptive immunity is enhanced. More specifically, enhanced expression of cytokines INF-γ and IL-2 suggests that major Th1 cell immunity was significantly improved by the presence of PIKA adjuvant. The activity of the cellular immune response is a key feature essential for treating intracellular viral, bacterial, and parasitic infections, and in particular an important factor for the development of therapeutic vaccines.

Furthermore, compositions containing PIKA stimulated INF-γ production by CD4 ⁺ T cells (Examples 1.3, 1.4, 1.5, and 1.6). This feature demonstrates that PIKA enhances the host's adaptive immune response.

  The observed increase in serum antibodies indicates that PIKA adjuvant induces an effective humoral response. By adding PIKA to the immunogenic composition, an increase in the IgG titer, a specific antibody, was observed (Examples 1.1, 1.2, 1.4, 1.5, 1.6, 2 3, 5, and 6).

  PIKA adjuvants include inactivated antigens (Examples 1.2, 1.6, 2, 3, 4, and 6), peptide antigens (Example 1.3), and recombinant antigens (Examples 1.1, 1.. When combined with 4, 1.5, 5, and 7) enhances the immune response in the host.

  A particular feature of PIKA adjuvants is to provide adequate protection to reduce and / or eradicate infection in the host and / or reduce the risk of disease symptoms resulting from infection by pathogens. . VAXIGRIP (Sanofi Pasteur) used as an antigen in Examples 1.2 and 6 is a human influenza vaccine itself and is considered to be sufficient to provide protection against acute influenza infection. Induces immune activity. Addition of PIKA to VAXIGRIP further enhanced the immune response, as demonstrated by the extent of beneficial cytokines (IL-2, INF-γ, and IL-4) and specific IgG expressed by the immune system.

  To further demonstrate the protective properties of PIKA, 24 10-day-old chickens were inoculated with a composition comprising PIKA and inactivated avian influenza antigens including H5N1 and H9N2 strains (Example 3). Subsequently, these chickens were inoculated with H5N1 live virus and observed for a period of 2 weeks. At the end of this plan, PIKA / antigen composition was not compared to PIKA / antigen composition, compared to only 17% of 24 control chickens that were not pre-inoculated with PIKA / antigen and inoculated with live virus. The survival rate of chickens inoculated was 83%.

  In a related experiment to demonstrate the enhanced therapeutic properties of PIKA adjuvant, Balb / c mice were inoculated with a wild strain of rabies virus (Example 4). After infection, three different groups of animals were vaccinated with treatment regimens with different rabies vaccines. The survival rate of the mice group inoculated with a combination of inactivated rabies antigen purified from hamster kidney cells and PIKA reached 80%. The survival rate of the second group of mice inoculated with rabies antigen purified from hamster kidney cells and alum adjuvant was 15%. In addition, the third group of mice administered “Verorab” from Sanofi-Aventis had a survival rate of 20%. Verolab is an inactivated rabies vaccine derived from Vero cells.

  In further demonstration of the properties of the PIKA adjuvant, in Example 7, the presence of PIKA simultaneously with the HBsAg adw type enhances the production of specific IgG in mouse serum (Table 26 FIG. 35), and the titer of IgG2a Has been demonstrated to be more significantly enhanced (Table 27, Figure 36). The conclusion from this observation is that PIKA enhances the therapeutic immune response, particularly an immune response biased toward Th1.

Related experiments (Example 7) show that the presence of PIKA in vaccine formulations containing HBsAG adw type enhances interferon-γ production by splenocytes stimulated with CD8 T cell peptide epitopes. It was. This result demonstrates that PIKA induces a CD8 ⁺ T cell immune response (Table 28 FIG. 37).

  Furthermore, (Example 7) The presence of PIKA in a vaccine preparation containing HBsAg enhances the production of interferon-γ by splenocytes cultured in vitro with 2 ug / ml of CD8 T cell peptide epitope for 6 days. It has been shown. This result demonstrates that PIKA induces a central memory T cell response.

<Additional features>
In a further embodiment, the subject immunogenic composition is further characterized by the relative presence of the PIKA adjuvant and the antigen or antigens. In this case, the presence is measured in terms of one or more characteristics among quantity, concentration, volume, number of molecules, or other certified metrics.

  In a related embodiment, the subject immunogenic composition comprises a polynucleotide adjuvant composition and one antigen or multiple antigens, wherein the presence of adjuvant and antigen with respect to molecular weight or number is less than 1: 1000 A ratio lower than 900, a ratio lower than 1: 800, a ratio lower than 1: 700, a ratio lower than 1: 500, a ratio lower than 1: 400, a ratio lower than 1: 300, a ratio lower than 1: 200, 1: Ratio lower than 100, ratio lower than 1:50, ratio lower than 1:10, ratio lower than 1: 5, ratio lower than 1: 2, ratio higher than about 1: 1, 2: 1, higher than 5: 1 Ratio, ratio higher than 10: 1, ratio higher than 50: 1, ratio higher than 100: 1, ratio higher than 200: 1, ratio higher than 300: 1, ratio higher than 400: 1, ratio higher than 500: 1 A ratio higher than 600: 1, 00: higher than 1 ratio, 800: greater than 1 ratio, 900: greater than 1 ratio, 1000: higher specific than 1.

  In a further related embodiment, the subject immunogenic composition is characterized in terms of dosage. Dosage means the amount of vaccine administered to induce an appropriate and beneficial immune response, or the possibility of inducing unintended side effects from the minimum dose required to elicit an immune response The range of doses that can be administered is up to the maximum dose at which no additional beneficial response is medically proven.

  In an embodiment of particular interest, the immunogenic composition comprises a polynucleotide adjuvant composition and an antigen, and the presence of the antigen in a unit dose is provided in the following amounts. That is, more than 0.1 ug, more than 0.5 ug, more than 0.001 mg, more than 0.005 mg, more than 0.01 mg, more than 0.025 mg, more than 0.05 mg , More than 0.075 mg, 0.1 mg, more than 0.25 mg, more than 0.5 mg, more than 1.2 mg, more than 1.4 mg, more than 1.6 mg, 1.8 mg Greater than, greater than 2.0 mg, greater than 2.5 mg, greater than 3 mg, greater than 3.5 mg, greater than 4 mg, greater than 5 mg, greater than 6 mg, greater than 7 mg, An amount greater than 8 mg, an amount greater than 9 mg, an amount greater than 10 mg, an amount greater than 15 mg, an amount greater than 20 mg, an amount greater than 25 mg, or an amount greater than 50 mg.

  The appropriate antigen amount and the appropriate ratio of antigen to PIKA adjuvant can be determined by standard studies including observation of antibody titers and other immunogenic responses in the host.

<Antigen>
In a particularly noted embodiment, the present invention provides a polynucleotide adjuvant composition with an antigen or vaccine. In this case, the source of the antigen is a human antigen, an animal antigen, a plant antigen, any virus, one or more substances from bacteria, fungi, or parasite-derived infectious substances, cancer antigens, allergies Sexual substances and other antigens that develop autoimmune diseases.

  In certain embodiments, the antigen may be derived from a crude or purified natural source, the original live form of the antigen, or a post-kill antigen, or an inactive antigen, or a truncated antigen, or an attenuated antigen, or Transformable antigen to irreversible form, or detoxified antigen, or mutant antigen to nontoxic form, or filtered antigen, or purified antigen may be used.

  In some embodiments, the antigen is an isolated microorganism antigen such as, for example, a viral antigen, a bacterial antigen, a fungal antigen, an allergic antigen, a cancer antigen, or an autoimmune antigen. In other embodiments, the antigen is an inactivated complete antigen. Methods for inactivating complete antigens are well known techniques. That is, some known methods can be used to inactivate an antigen and can be selected appropriately depending on the type of antigen. Such antigen inactivation methods include, for example, photoreactive compounds; oxidizing agents; irradiation (for example, radiation irradiation such as ultraviolet rays and γ-ray irradiation); a combination of riboflavin and ultraviolet irradiation; solvent-surfactant treatment ( For example, treatment of organic solvent tri-N-butyl-phosphate with a surfactant such as Tween 80); polyethylene glycol treatment; pasteurization (heat treatment); and low pH treatment; mild enzyme with pepsin or trypsin Treatment; treatment with methylene blue (MB); treatment with dimethylmethylene blue (DMMB) and visible light; treatment with psoralen derivative S-59 and ultraviolet A irradiation; and the like.

  In a related embodiment of interest, the antigen may be synthesized by solid phase synthesis or obtained by genetic engineering or so as to mimic the immunogenic characteristics of the pathogen. It may be manufactured artificially by another method.

  Polypeptide antigens may be isolated from natural sources using standard protein purification methods that are known in the art. Protein purification methods include liquid chromatography (eg, high performance liquid chromatography, high performance protein liquid chromatography, etc.), size exclusion chromatography, gel electrophoresis (including one-dimensional gel electrophoresis, two-dimensional gel electrophoresis), affinity Including but not limited to chromatography or other purification techniques. Solid phase peptide synthesis techniques may be employed and such techniques are known to those skilled in the art. See Jones, “The Chemical Synthesis of Petides” (Clarendon Press, Oxford) (1994). In general, in such methods, peptides are produced by sequentially adding active monomer units to a solid phase bound extended peptide chain. Well established recombinant DNA technology may be employed for the production of polypeptides. Such recombinant DNA techniques include, for example, expression constructs comprising a nucleotide sequence encoding a polypeptide in a suitable host cell (e.g., a eukaryotic host cell that grows in vitro cell culture as a unicellular entity, e.g., Yeast cells, insect cells, mammalian cells, etc.), or prokaryotic cells (eg, growing in in vitro cell culture), including, but not limited to, methods for creating genetically modified host cells . That is, under appropriate culture conditions, proteins are produced by host cells that have been modified by genetic engineering.

  In some embodiments, the antigen is, for example, about 25% to 50% pure, about 50% to about 75% pure, about 75% to about 85% pure, about 85% to about 90% pure, about 90% A purified antigen having a purity of about 95% purity, about 95% to about 98% purity, about 98% to about 99% purity, and higher than 99% purity.

  The antigen may be acellular, capsule, infectious clone, replicon, vectored, microencapsulated, monovalent, divalent, or multivalent.

  The polynucleotide adjuvant composition of the present invention can also be used to enhance an immune response against an antigen produced by a DNA vaccine and / or a DNA-expressed protein. The DNA sequence encoding the antigen in these vaccines may be intact or housed in a delivery system such as a liposome.

  In one aspect of particular interest, the subject immunogenic composition can be characterized by the selection of the antigen or antigens used in combination with the PIKA adjuvant.

  More specifically, the present invention provides immunogenic compositions and methods of use. In this case, the immunogenic composition comprises a PIKA adjuvant together with a viral antigen. Exemplary antigens include, but are not limited to, one or more viral antigens listed in Table 1.

  More specifically, the present invention provides immunogenic compositions and methods of use. In this case, the immunogenic composition comprises a PIKA adjuvant together with a bacterial antigen. Exemplary antigens include, but are not limited to, one or more bacterial antigens listed in Table 2.

  More specifically, the present invention provides immunogenic compositions and methods of use. In this case, the immunogenic composition comprises a PIKA adjuvant together with a fungal antigen. Exemplary antigens include, but are not limited to, one or more fungal antigens listed in Table 3.

  More specifically, the present invention provides immunogenic compositions and methods of use. In this case, the immunogenic composition comprises a PIKA adjuvant together with a parasitic antigen. Exemplary antigens include, but are not limited to, one or more parasite antigens listed in Table 4.

  In related embodiments, the present invention provides immunogenic compositions and methods of use. In this case, the immunogenic composition comprises a PIKA adjuvant together with an allergic antigen (so-called allergen) or vaccine, and the origin of the antigen or vaccine is derived from or from a human or animal allergen Produced by imitating. Animal allergens include plants, animals, fungi, insects, cereals, drugs, dust, ticks and the like.

  Allergens include ambient air allergens; pollen of plants such as ragweed / hay fever; weed pollen allergens; grass pollen allergens; Johnson grass; tree pollen allergens; ryegrass; pI, Der f I, etc.); warehouse mite allergen; cedar pollen / hay fever; mold spore allergen; animal allergen (eg, allergens such as dogs, guinea pigs, hamsters, gerbils, rats, mice); Crustacean allergens; Peanut-like nut allergens; Citrus fruit allergens; Insect allergens; Toxins: (Hymenoptera, Hornets, Honeybees, Hornets, Hornets, Crabs); Insect allergens around Bacterial allergens such as streptococcal antigen; parasite allergens such as Ascaris antigen; viral antigens; fungal spores; drug allergens; antibiotics; penicillins and related compounds; other antibiotics; hormones (insulin) Complete proteins such as enzymes (streptokinase); all drugs and their metabolites that can act like incomplete antigens or haptens; industrial chemicals that act like haptens and can function as allergens and Metabolites (eg, acid anhydrides (such as trimellitic anhydride) and isocyanates (such as toluene diisocyanate)); flour (eg, allergens that cause bakery asthma), castor bean, coffee beans, and the above Occupational alleles like industrial chemicals Emissions; flea allergens; and, including human proteins in non-human animals, but not limited to.

  Allergens include cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide complexes, peptides and non-peptide mimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, and carbohydrates. Including, but not limited to.

  Examples of natural allergens, animal allergens, and plant allergens include proteins specific to the following genera: Canine (Canis familiaris); Dermatophagoides (eg Dermatophagoides farinae) )); Felics (Felis domesticus); Ambrosia (Ambrosia artemiisfolia); Lolium (eg, Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinosa) Birch (Betula) (Betula verrucosa); Quercus (Kuwe) Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (eg, Plantago lanceolata); Parietaria (eg, Parietaria officinalis or Parietaria judaica); Blattella (eg, Blattella germanica); Apis (eg, Apis rum) (Apis multiflorum); Cupressus (eg, Cupressus sempervirens, Cupressus arizonica, and Cupressus macrocarpa); Juniperus (eg, Uniperus)・ Sabionoi Death (Juniperus sabinoides), Uniperus virginiana, Juniperus communis, and Juniperus ashei; Thuya (eg, Thuya orientalis); Chamaecyparis (eg, Chamaecyparis obtusa); Cockroach (eg, Periplaneta americana); Camellia (eg, Agropyron repens); Rye Secale (eg, Secale cereale); Wheat (Triticum) (eg, Triticum aestivum); Dactylis (eg, Dactylis glomerata); Festuca (eg, Festuca elatior); Strawberry genus (Poa) (E.g., Poapratensis or Poa compressa); Avena (e.g., Avena sativa); Holcus (e.g., Holcus lanatus)); Anthoxanthum (eg, Anthoxanthum odoratum); Arrhenatherum (eg, Arrhenatherum elatius); Agrostis (eg, Agrostis alba); Phleum pratense); Phalaris (eg, Phalaris arundinacea); Paspalum (eg, Paspalum notatum); Sorghum (eg, Sorghum) (Sorghum halepensis)); and the genus Bromu s) (eg, but not limited to, Bromus inermis).

  In related embodiments, the present invention provides polynucleotide adjuvant compositions and methods of use where the immunogenic composition comprises a PIKA adjuvant together with an autoimmune antigen or vaccine.

  In related embodiments, the present invention provides immunogenic compositions and methods of use. In this case, the immunogenic composition contains only the PIKA adjuvant or a cancer antigen. Exemplary antigens include, but are not limited to, one or more cancer antigens listed in Table 5.

  In related embodiments, the origin of the cancer antigen may be as follows. That is, 1) Viral proteins such as hepatitis B virus (HBV), Epstein-Barr virus (EBV), and human papilloma virus (HPV) are respectively hepatocellular carcinoma, lymphoma, and child It is important for the development of cervical cancer. 2). Whole cancer cells (which may be inactivated) and / or unpurified and / or semi-purified extracts of these cells; 3). Tumor-associated antigens (TAAs) such as tumor-specific carcinogenic proteins, glycosylated proteins, gangliosides, glycolipids, mucins, peptides, carbohydrates and anti-idiotype monoclonal antibodies.

  In related embodiments, an immunogenic composition comprising a polynucleotide adjuvant is used to prevent further growth of existing cancer, prevent recurrence of a treated cancer, or remove cancer cells that were not killed by previous therapy. It may be used to treat cancer tumors. This treatment may occur prior to, in conjunction with, or after other treatments provided to the individual. Therefore, it may be part of a comprehensive combination treatment to treat cancer.

  In a related embodiment, the cancer vaccine provides a treatment that can induce a tumor-specific immune response against both early and metastatic tumors. In addition, the induction of strong immunity can lead to the establishment of immune memory. As a result, tumor recurrence can be reduced or prevented. The cancer vaccine may induce a specific antibody against a tumor-associated surface antigen, and more preferably induces a cellular immune response that is biased to a Th1 immune response as much as possible.

  Any of a variety of known tumor-specific antigens or tumor-associated antigens (TAA) can be included in a subject immunogenic composition. All TAAs are not necessarily required but may be used. Alternatively, a portion of TAA, such as an epitope, may be used. Tumor associated antigens (or fragments containing epitopes within tumor associated antigens) may be used for YFV, and tumor associated antigens are MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2. , High molecular weight melanoma associated antigen MAA, GD2, carcinoembryonic antigen (CEA), TAG-72, ovarian associated antigens OV-TL3 and MOV18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, CA-50 , CA-19-9, renal tumor associated antigen G250, EGP-40 (also known as EpCAM), S100 (malignant melanoma associated antigen), p53, and p21ras. Synthetic analogs of any TAA (or epitopes thereof) including any of the aforementioned TAAs may be used. Further, combinations of one or more TAAs (or epitopes thereof) may be included in the composition.

  In some embodiments, a subject immunogenic composition comprises a polynucleotide adjuvant and at least two different antigens. For example, in some embodiments, a subject immunogenic composition comprises two antigens, three antigens, four antigens, five antigens, or more than five antigens.

<Additional substances>
In some embodiments, a subject immunogenic composition includes one or more additional substances, such as immunostimulants, carriers, etc., in addition to the PIKA adjuvant and antigen.

  In particularly noteworthy embodiments, the present invention provides immunogenic compositions and methods of use thereof. In this case, the immunogenic composition includes a PIKA adjuvant, antigen or vaccine along with other immunostimulatory substances including an adjuvant, suitable immunostimulatory substances being an aluminum composition such as aluminum hydroxide; oil-in-water An oil-in-water emulsion composition or an emulsion containing an immunogenic substance containing Freund's complete adjuvant; an oil-in-water emulsion containing a dried, heat-killed Mycobacterium tuberculosis organism; Complete adjuvant; emulsion composition containing mycobacterial cell walls; emulsion containing squalene (MF-59); lipid A derivative, a detoxified endotoxin containing monophosphoryl lipid A microorganism (MPL); hapten; nitrocellulose-absorbing protein; Particulate immunostimulant isolated from Quillaja saponaria skin, eg QS A saponin containing 1; an endogenous human immunostimulant; a microbial-derived adjuvant containing an unmethylated CpG dinucleotide; an oligodeoxynucleotide (eg a synthetic oligonucleotide) containing an unmethylated CpG dinucleotide; a liposome (eg a phospholipid of Liposomes made from biodegradable materials such as: biodegradable polymer microspheres (eg, polylactic-co-glycolic acid (PLGA), polyphosphazenes, and polyanhydrides). Microspheres made from various polymers such as); interleukin-2; Bacille Calmette Guerin; granulocyte monocyte-colony stimulating factor; Montanide ISA-51; keyhole limpet hemocyanin; DNA; protein; capsule antigen; Epidemic stimulating complex (ISCOM's); cholera toxin, cholera toxin derivative; closed zone toxin; Escherichia coli burning enterotoxin; unstable toxin, unstable toxin derivative; pertussis toxin, pertussis toxin derivative; muramyl dipeptide derivative; Including, but not limited to, a seppic series of monotanide adjubant; poly-di (carboxylatophenoky) phosphazene, and leishmania elongation factor.

  When the subject immunogenic composition is administered in conjunction with other adjuvants, the polynucleotide adjuvant may be before administration of the other adjuvant and / or after administration of the other adjuvant and / or of other adjuvant. It can be administered simultaneously with administration. For example, the polynucleotide adjuvant may be administered in conjunction with the initial administration of the antigen, followed by a booster vaccine comprising one or both of the adjuvants. Alternatively, the initial vaccine may exclude the polynucleotide adjuvant, but the immunogenic material containing the polynucleotide adjuvant is then administered to the patient.

  In certain embodiments, a subject immunogenic composition is a cytokine or other costimulatory molecule, such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10. , IL-12, and IL-15.

  In a related embodiment, the present invention provides an immunogenic substance comprising a suitable carrier in addition to a PIKA adjuvant, one antigenic substance or a plurality of antigenic substances. Carriers include, for example, oil-in-water emulsions, suspensions, lipid vesicles, aluminum salts, cochleates, ISCOMs, liposomes, live bacterial vectors, live viral vectors, microviruses. Spheres, nucleic acid vaccines, polymers, polymer rings, sodium fluoride, genetically modified plants, virosomes, virus-like microparticles, and other delivery vesicles known in the art.

  The polynucleotide adjuvant may be administered directly to the subject or may be administered in combination with a delivery complex. In this case, the transport complex is a substance associated with the targeting means, for example a molecule that provides high affinity binding to the target cell, such as the surface of the dendritic cell, and / or increases intracellular uptake by the target cell. Is a molecule. Typical delivery complexes are recognized by sterols (eg, cholesterol), lipids (eg, cationic lipids, virosomes, or liposomes), or target cell specific binders (eg, target cell specific receptors). A nucleic acid-carrying acid associated with a ligand), but is not limited thereto. Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to being taken up by target cells. However, the complex may be cleavable under appropriate conditions within the cell.

  In one noted embodiment, a composition comprising a PIKA adjuvant does not comprise poly-L-lysine or a derivative thereof.

<Kit>
In certain embodiments, the present invention provides kits comprising a subject immunogenic composition. In certain embodiments, the present invention provides kits comprising PIKA adjuvant and antigen in separate formulations.

  In a related embodiment, the present invention provides a kit comprising a polynucleotide adjuvant and an immunogenic compound. In this case, the immunogenic substance is an antigen.

  In some embodiments, the subject kit comprises the subject immunogenic composition in a sterile liquid formulation (eg, an aqueous formulation). In this case, the formulation is sterile and is provided in a sterile container, sterile vial, or sterile syringe.

  In some embodiments, a subject kit includes a subject immunogenic composition formulated for injection. In some embodiments, a subject kit includes a subject immunogenic composition in a sterile liquid formulation placed in a sterile syringe; and a needle. In some examples, a subject kit includes a subject immunogenic composition in a unit dosage (eg, a single dose) sterile liquid formulation placed in a sterile syringe; and a needle .

  In some embodiments, a subject kit includes a container comprising a lyophilized subject immunogenic composition in a sterile container; and a sterile solution for reconstitution of the lyophilized composition. In some embodiments, the kit further comprises instructions for reconstituting the lyophilized composition.

  In some embodiments, the subject kit comprises rectal administration, vaginal administration, nasal administration, oral administration (including inhalation), opsamalically administration, topical administration, pulmonary administration, ocular administration, or skin. It includes an immunogenic composition formulated for administration, and a suitable delivery device such as an aspirator, suppository, applicator or the like.

  The subject kit in some embodiments will further comprise instructions for use including, for example, dosage and frequency of administration. In some embodiments, the instructions for use are printed directly on the kit. In other embodiments, the instructions for use are printed matter provided as a written book. Instructions for use can also be provided electronically on other media, eg, audio cassettes, audio tapes, compact discs, digital versatile discs, etc., eg, in digital or analog form.

<Formulation>
A subject immunogenic composition is provided in any of a variety of formulations. For example, a subject immunogenic composition can be an injectable substance, a dry powder, a solution (eg, an aqueous solution or saline), or a suspension, cream, emulsion, tablet, coated tablet, microcapsule, suppository, instillation , Pills, granules, dragees, capsules, gels, syrups or slurries. The preparation of the desired immunogenic composition formulation is generally described in Vaccine 4 ^th Edition (Stanley A Plotkin et al., WB Saunders Company; 4th edition 2003). Suitable formulations, "Remington: The Science and Practice of Pharmacy" ^{((2000) A.Gennaro, 20 th} edition, Lippincott, Williams, &Wilkins); Pharmaceutical Dosage Forms and Drug Delivery Systems ((1999) HC Ansel et al , eds., 7 ^th ed., Lippincott, Williams, &Wilkins); and Handbook of Pharmaceutical Excipients ((2000) AH Kibbe et al., eds., 3 ^rd ed. Amer. Pharmaceutical Assoc.); Methods in Molecular Medicine, Vol. 87: Vaccine Protocols, 2nd edition ((2003), Humana Press); Mucosal Vaccines ((1996), Kiyono et al., Eds., Academic Press); and Vaccine Adjuvants: Preparation Methods and Research Protocols ((2000) DT O'Hagan, Humana Press).

  The subject immunogenic composition may be contained in microcapsules, may be contained in cocreatate, may be coated on tiny gold particles, or may be contained in liposomes. Alternatively, it may be a spray aerosol, may be a pill implanted under the skin, or may be dried on a sharp object (eg, a needle) for damaging the skin.

  In further embodiments, the subject immunogenic material may be delivered alone or in combination with a dispersion. In some embodiments, the dispersion is selected from the group consisting of, for example, a polymer complex based system, a nanocapsule based system, a microsphere based system, a bead based system, and a lipid based system. Lipid-based systems optionally include oil-in-water emulsions, micelles, mixed micelles, or liposomes.

  In certain embodiments, a subject immunogenic composition comprising a PIKA adjuvant is in the form of a pharmaceutically acceptable solution, usually a pharmaceutically acceptable concentration of salt, buffer, preservative, compatible carrier. , Adjuvants, and optionally other therapeutic ingredients. The composition may contain additives such as tablet disintegrants, binders, coatings, swelling agents, lubricants, flavoring agents, sweeteners, or solubilizers.

  In certain embodiments, a subject immunogenic composition comprising a PIKA adjuvant is administered in a pharmaceutically acceptable intact form or in the form of a pharmaceutically acceptable salt.

  The immunogenic composition of the present invention is a capsule, solution, droplet, emulsion, suspension, elixir, cream, suppository, gel, soft capsule, spray, inhaled antigen, aerosol, powder, tablet, coated tablet, troche , As microcapsules, suppositories, dragees, syrups, slurries, granules, enemas, or pills, in both sterile and non-sterile forms. Any inert carrier may be used. For example, the compounds used in the methods of the invention in saline or phosphate buffered saline covered with gelatin capsules or microcapsules, preservatives, high pressure gas, or media to aid administration, or any carrier. Any carrier having dissolution characteristics suitable for use in the inventive method can be used.

  In certain embodiments, PIKA adjuvant compositions and immunogenic compositions comprising PIKA adjuvants and antigenic compounds are lyophilized and stored in solid form for long term stability. Freeze-drying methods are known to those skilled in the art.

  In one aspect of interest, the invention provides an adjuvant composition or immunogenic composition, wherein the adjuvant composition included in the immunogenic composition or immunogenic composition is a solid or liquid, or Present in a solution or suspension or in an emulsion.

  The subject immunogenic composition is administered to an individual by a drug delivery system method for inhalation administration methods (oral administration, intratracheal administration, nasal administration). Thus, the subject immunogenic composition may be formulated in a form suitable for administration by inhalation. The drug delivery system is a delivery system suitable for the treatment of the respiratory system by locally administering the target bacterial composition to the mucosal lining of the bronchi. The invention can utilize a system that relies on compressed gas powder to fire bacteria from a container. Aerosols or pressurized containers can be used for this purpose.

  As used herein, the term “aerosol” is used in its conventional sense to represent very fine liquid or solid particles that are carried to a treatment application site by high pressure gas under pressure. When an aerosol pharmaceutical is used in the present invention, the aerosol comprises an immunogenic composition that is dissolved, suspended, or suspended in a mixture of a flowable carrier and a high pressure gas. It can be emulsified. Aerosols can be prepared in solution, suspension, emulsion, powder, or semi-solid form. The aerosol used in the present invention is intended for administration as fine solid particles or liquid vapor via the subject's airways. Various types of high pressure gas known to those skilled in the art can be utilized. Examples of suitable high pressure gases include, but are not limited to, hydrocarbons or other suitable gases. In the case of a pressurized aerosol, the dosage unit can be determined by providing a value for carrying the metered amount.

  There are several different types of inhalation methods that can be used in conjunction with the present invention. The subject immunogenic composition can be formulated in basically three different types of inhalation formulations. First, the subject immunogenic composition can be formulated with a low boiling high pressure gas. Such formulations are generally administered by conventional metered dose inhalers (MDI's). However, as discussed in US Pat. Nos. 5,404,871 and 5,542,410, conventional MDI's can be obtained by using techniques to measure a subject's inhalation volume and flow rate. Modifications can be made to increase the ability to achieve reproducible dosing.

  Alternatively, the subject immunogenic compositions can be formulated in aqueous or ethanol solutions and delivered by conventional nebulizers. In some embodiments, such solution formulations are described in US Pat. Nos. 5,497,763; 5,544,646; 5,718,222; and 5,660,166. Aerosolized using devices and systems as disclosed.

  Further, the subject immunogenic composition is formulated into a dry powder formulation. Such formulations can be administered by simple aspiration of the dry powder formulation after creating a powder aerosol vapor. Techniques for accomplishing the above are described in US Pat. No. 5,775,320 and US Pat. No. 5,740,749. Formulations suitable for intranasal administration include nasal sprays, nasal drops, aerosol formulations and the like.

  In some embodiments, a subject immunogenic composition is formulated for sustained release (eg, a controlled release formulation). For example, in some embodiments, a subject immunogenic composition is formulated in a pill or cylinder and implanted intramuscularly or subcutaneously as a reservoir injection or implant. Such implants generally use known inert materials such as biodegradable polymers. Injectable depot forms are made by forming microencapsulated matrices of the subject immunogenic compositions in biodegradable polymers such as polylactide-polyglycolide. Examples of other suitable biodegradable polymers include polyorthoesters and polyanhydrides. Reservoired injection formulations are also prepared by encapsulating the composition in liposomes or microemulsions that are compatible with living tissue. Examples of delivery systems include: polymer-based systems, microcapsules, lipids, hydrogel release systems, sylastic systems, peptide systems, peptide-based systems, wax processing, compressed tablets, partial fusion implants Other sustained release forms known to those skilled in the art are also included.

<Method>
In one aspect of particular interest, the present invention provides a method of inducing and / or enhancing an immune response to an antigenic compound. This method involves administration of a subject immunogenic composition to a host. In some embodiments, the host is a human. In other embodiments, the host is a non-human animal, such as a non-human mammal, a bird, and the like.

  In certain embodiments, the polynucleotide adjuvant composition can be used in the context of a vaccine. Optionally, the vaccine composition includes an additional adjuvant. The types of vaccines included are anti-infective, anti-cancer, anti-allergic, and anti-autoimmune diseases.

  Furthermore, the present invention provides a method for enhancing an immune response to an antigenic compound by administering a subject immunogenic composition to a host. The host can be a human or non-human animal.

  In certain embodiments, the adjuvant is administered with the antigen. In further embodiments, the adjuvant is administered before or after administration of the antigen.

  The subject immunogenic composition is, in some embodiments, delivered parenterally by injection, such as intramuscular injection, intraperitoneal injection, intravenous injection, or intradermal injection. In other embodiments, the immunogenic composition is administered transdermally by a method other than injection, eg, a method that does not damage the epithelial barrier by mechanical means. In other embodiments, the immunogenic composition is delivered from the rectum, from the vagina, from the nose, from the mouth (including inhalation), to the opsammary, from the eyeball, topically, from the lungs, or from the skin. The

  A subject may be exposed to an antigen by environmental contact and, as a result, may be at risk of developing an allergic reaction, infection, autoimmune disease, or cancer, for example. In other embodiments, the subject becomes pre-exposed to an antigen through environmental contact, resulting in, for example, an infection, an autoimmune disease, cancer, or allergy.

  In certain embodiments, when the method of administering an immunogenic composition comprising a polynucleotide adjuvant is for the treatment of cancer tumors, delivery is by direct injection into the tumor or by injection near the tumor . In some embodiments, the immunogenic composition is uniformly delivered throughout the tumor or throughout the tumor to improve biodistribution and enhance therapeutic efficacy.

  For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. Should. These special aqueous solutions are particularly suitable for intravenous and intraperitoneal administration. In this regard, one of ordinary skill in the art will know the sterile aqueous solvents that can be used in light of the present disclosure. Typical injectables used in the present invention were added with dispersants and / or preservatives and edible oil, mineral oil, cod liver oil, squalene, monoglycerol, diglycerol, triglycerol, and mixtures thereof Or it contains a buffer that has not been added.

  A subject immunogenic composition is administered in an “effective amount”. An effective amount is that amount of a subject immunogenic composition that is effective to elicit, induce or enhance an immune response in the chosen route of administration. In some embodiments, the immune response is elicited against an antigen produced by the pathogenic microorganism. In some embodiments, the amount of the subject immunogenic composition is effective in limiting infection by the pathogenic organism and / or eradicating the infection and / or reducing symptoms associated with the infection. is there.

  For example, in some embodiments, administration of a subject immunogenic composition to an individual is effective in treating an infection. In this case, treatment of an infectious disease means reducing the number of pathogens in an individual (eg, reducing viral load, reducing bacterial load, reducing protozoa, reducing helminth), and / or infection. Includes one or more of reducing disease-related parameters. Infectious disease-related parameters include a reduction in the amount of product (eg, toxins, antigens, etc.) produced by the pathogen and a reduction in unwanted physiological responses to the pathogen (eg, fever, tissue edema, etc.), It is not limited to these.

  The exact amount of such composition required will vary from subject to subject and will vary from subject to subject, age, weight and general condition, disease to be treated or prevented, infection or severity of disease, detailed compounds used As well as the method of administration thereof. Using only the routine experiment indicated herein, the appropriate amount can be determined by one of the techniques common in the art. Following the initial administration, the subject may receive one or several booster immunizations with sufficient duration.

  In some embodiments, the subject immunogenic composition is administered continuously. In these embodiments, a first dose of the subject immunogenic composition may be administered in response to the results of vaccine administration. After exposure to the first dose and the individual is immunologically ready, a second dose of the subject immunogenic composition is administered to the individual. Additional antigen doses may be administered days, weeks, or months after the initial dose, depending on patient response and condition. For example, the booster dose is from about 2 days to about 12 months from the first time, for example, from about 2 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, It is administered after about 4 weeks to about 8 weeks, about 8 weeks to about 6 months, and about 6 months to about 12 months. The present invention may be, for example, 3rd, 4th, 5th, 6th, or subsequent dose, 3rd, 4th, 5th, 6th, or Further use of subsequent boosts is further contemplated.

  In certain embodiments, the method of administration may include alternative route combinations. For example, systemic administration (eg, peritoneal, intramuscular, subcutaneous, or intradermal) may be followed by mucosal delivery (eg, nasal, inhalation). Or vice versa.

  In certain embodiments, the polynucleotide adjuvant may be administered together with either the initial dose or subsequent dose of antigen to be administered, or may be administered with all doses administered to the patient. At least one of the doses administered as part of the overall procedure will include a PIKA adjuvant.

  In certain embodiments, the composition of the administered immunogenic composition may differ between the initial administration and the booster and / or between the booster doses. As an example, the initial dose includes a DNA vaccine, but the booster dose may be in the form of a recombinant protein vaccine. At least one of the doses administered as part of the overall procedure will include a PIKA adjuvant.

  Whether an antibody response to an antigen is induced or enhanced in an individual is readily demonstrated using standard tests. Immunological tests such as, for example, enzyme immunoassays (ELISA), radioimmunoassays (RIA), immunoprecipitation tests, and protein blot tests (“Western” blots); and neutralization tests (eg, in vitro or in vitro) neutralization of the infectivity of the virus in vivo); and these tests are performed for antibodies specific for bacterial antigens in body fluids or other biological samples, such as serum, secretions, or other fluids of individuals. Can be used to detect presence.

  Whether a CD4 immune response to an antigen was induced in an individual is readily demonstrated using standard tests. For example, fluorescence activated cell sorting (FACS) (see, eg, Waldrop et al. (1997) J. Clin. Invest. 99: 1739-1750); intracellular cytokine tests that detect cytokine production following antigen stimulation (eg, Suni et al. (1998) J. Immunol. Methods 212: 89-98; Nomura et al. (2000) Cytometry 40: 60-68; Ghanekar et al. (2001) Clin. Diagnostics Lab Immunol. 8: 628- 631); MHC-peptide multimer staining tests, eg the use of soluble MHC Class II / peptide multimers with a detectable label (eg fluorescent label) (eg Bill and Kotzin (2002) Arthritis Res. 4: 261-265; Altman et al. (1996) Science 274: 94-96; and Murali-Krishna et al. (1998) Immunity 8: 177-187); enzyme-linked immunospot (ELISPOT) Tests (eg Hutchings et al. (1989) J. Immunol. Methods 120: 1-8; and Czerkinsky et al. (1983) J. Immunol. Methods 65:10 9-121). As one non-limiting example of an intracellular cytokine test, whole blood is stimulated with an antigen and a costimulatory antibody (eg, anti-CD28, anti-CD49d) for 2 hours or longer; Brefeldin A is added to inhibit cytokine secretion The cells are then FACS analyzed using fluorescently labeled antibodies against CD4 and fluorescently labeled antibodies against cytokines such as TNF-a, INF-γ and IL-2.

Whether an antigen-specific CD8 (eg, cytotoxic T cell; so-called CTL) response is induced against an antigen (eg, a pathogen) is demonstrated using any of a number of well-known tests. can do. Testing includes, but is not limited to, measurement of specific lysis by CTL in target cells that express the antigen on the cell surface. The target cells incorporate a detectable label that is released from the target cells upon lysis and can be measured using, for example, a ⁵¹ Cr-release test; a cytokine test based on lanthanide fluorescence.

<Subject suitable for treatment>
Subjects suitable for treatment with methods of inducing an immune response to bacterial pathogens and methods of treating or preventing infection of bacterial pathogens are individuals infected with pathogenic microorganisms; Including individuals who have not yet been infected; and individuals who are at risk of being infected with a pathogenic microorganism but have not yet been infected. Suitable subjects include infants, children, adolescents, and adults.

  Subjects suitable for treatment with methods of inducing an immune response against bacterial pathogens and methods of treating or limiting infection with bacterial pathogens include populations targeted by pediatrics. For example, individuals between about 1 and about 17 years old, such as infants (eg, about 1 month to about 1 year); children (eg, about 1 to about 12 years); and young people (eg, about 13 About 17 years old).

  Subjects suitable for treatment with methods of inducing an immune response against bacterial pathogens and methods of treating or limiting infection of bacterial pathogens include neonates. For example, individuals from 1 day to about 14 days of age (eg, human newborns), for example, from about 1 day to about 2 days, from about 2 days to about 10 days, from about 10 days to about 14 days. It is an individual of age.

  In certain embodiments, the subject is a human child about 10 years of age or younger, eg, about 5 years of age or younger. The immunogenic composition is then administered at any one or more of the following times: Specifically, 2 weeks after birth, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months Or 21 months, 2 years old, 3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, or 10 years old. In some embodiments, a subject immunogenic composition is administered to individuals ranging from about 6 months of age to about 6 years of age. In this case, the individual receives an initial dose at about 6 months of age and receives subsequent booster doses, for example, 2 to 3 subsequent booster doses, for example at 2 years, 4 years and 6 years. .

  In certain embodiments, the subject is a human adult about 17 to 49 years old. In some embodiments, the subject is an elderly human being 50 to 65 years old, 65 to 75 years old, 75 to 85 years old, 85 years old or older.

  In some embodiments, the subject immunogenic composition is in contact with an actual or potential source of bacterial pathogens (eg, contact is found or suspected of being contacted). Soon after being administered to an individual, eg, an individual infected with or suspected of being infected with a bacterial pathogen. For example, in some embodiments, a subject immunogenic composition is within about 1 hour, within about 2 hours, about 5 hours after contact with an individual infected with or suspected of being infected with a bacterial pathogen. Within 8 hours, within 8 hours, within 12 hours, within 18 hours, within 24 hours, within 2 days, within 4 days, within 7 days, within 2 weeks or within 1 month Be administered.

  In some embodiments, a subject immunogenic composition is provided to individuals who are known or suspected to be carriers or bacterial pathogens, regardless of whether the individual is exhibiting symptoms of infection. Be administered.

Subjects suitable for treatment with methods of inducing an immune response against bacterial pathogens and methods of treating or limiting infection of bacterial pathogens are individuals who are deficient in CD4 ⁺ T cells (“CD4 ⁺ -deficient”). “Individuals”, for example, individuals with lower numbers of functional CD4 + T lymphocytes than normal. As used herein, the term “normal individuals” refers to individuals who have a level and function of CD4 ⁺ T lymphocytes within the normal range within the population. In humans, usually, 1 mm 600 to 1500 CD4 ⁺ T lymphocytes per ³ blood is within the range. CD4 ⁺ -deficient individuals include individuals with acquired or primary immune deficiency. Acquired primary immunodeficiency may be temporary CD4 ⁺ deficiency due to radiation therapy or chemotherapy.

Individuals who are healthy and have an intact immune system but are at risk of becoming CD4 ⁺ deficient (an “at risk” individual) are also optimal for treatment using the methods of the invention. Individuals at risk include, but are not limited to, individuals that are more likely to become CD4 ⁺ deficient than the general population. Individuals at risk of becoming CD4 ⁺ deficient are individuals who are at risk of being infected with HIV due to sexual activity with individuals infected with HIV; drug users for intravenous injection; blood or blood products infected with HIV Or other individuals that may have been exposed to other HIV-contaminated body fluids; newborns through the birth canal of HIV-infected individuals; newborns breast-fed by HIV-infected mothers, but not limited to .

  Subjects suitable for treatment using targeted methods for cancer treatment include individuals infected with carcinogens, individuals who are likely to develop cancer but have not yet been diagnosed with cancer; and who are at risk of developing cancer. Includes individuals who have not yet been diagnosed with cancer. Suitable subjects include infants, children, adolescents, and adults.

  Subjects suitable for treatment using the subject method for cancer treatment are individuals diagnosed with cancer; for example, individuals who have been previously treated for cancer by chemotherapy or radiation therapy, and who have been previously treated Individuals who have been observed to have recurrence of cancer; and individuals who have undergone bone marrow transplantation or other organ transplants.

  Subjects suitable for treatment using the formulations and methods of the invention for allergy treatment include any individual diagnosed with allergy. Subjects susceptible to treatment with the methods and agents described herein include individuals who are known to have allergic hypersensitivity to one or more allergens. Subjects susceptible to treatment include individuals with any of the allergic diseases described above. Subjects at risk of having an allergic reaction to one or more allergens are also susceptible to treatment. Individuals who have failed treatment with standard therapies for the treatment of one or more allergic diseases are also optimal.

  Subjects suitable for treatment include individuals living in industrialized countries; individuals living in developing countries; individuals living in rural areas; individuals living in relatively isolated areas.

  The target population of the subject immunogenic composition will vary depending on the bacterial pathogen.

  The above disclosure primarily describes the present invention. The following examples will be helpful in understanding the present invention. These examples are described for illustrative purposes only and are not intended to limit the scope of the invention. If the method is suggested or provided by the surrounding environment, changes in form and replacement with equivalents are expected. Although special terms are used herein, such terms have been presented in a descriptive sense and not for purposes of limitation.

〔Example〕
[Example 1: PIKA induces specific immune responses with various antigens]
This example relates to the use of PIKA to elicit specific immune responses with various antigens in vivo. The study was performed in a series of independent experiments with a common protocol, each using a different antigen. Validated antigens include: hepatitis B surface antigen adw recombinant protein, inactivated and split influenza vaccine (VAXIGRIP provided by Sanofi Pasteur), synthetic HIV peptide antigen, herpes simplex Recombinant protein of virus type 2 gD antigen, recombinant anthrax protein protective antigen, inactivated avian influenza virus whole antigen H5N1 strain, and inactivated severe acute respiratory syndrome (SARS) virus whole Activated antigen.

  Protocols in individual experiments include compositions containing antigen only, compositions containing antigen and PIKA adjuvant (a heterogeneous composition of PIKA molecules that are mainly in the weight distribution range of about 66 kDa to 1200 kDa), PIKA only Or a control containing phosphate buffer (PBS) involves inoculating groups of Balb / c mice (3 mice per group).

  The actual dosage is defined for each antigen used. Mice were given the same booster vaccine 10-14 days after the first injection. Blood samples were collected 10-14 days after the booster injection, after which the mice were sacrificed and tissue samples were collected from the spleen. Results shown are the average of the test results for individual mice within each group.

After preparing a spleen cell suspension, cell suspension samples from each mouse were placed in 6-12 wells of ELISPOT plates and cultured. Each well of the ELISPOT plate contained 200 ul of a spleen cell suspension (approximately 2 × 10 ⁵ to 1 × 10 ⁶ cells / well) (see table below for details). For each mouse spleen cell sample to be cultured, half of the wells containing spleen cells are incubated with the culture medium, and the other half of the well contains two different concentrations of the particular antigen under evaluation. Stimulation was given using one of them. Prior to final preparation and reading using a standard ELISPOT plate reader, the plates are incubated at 37 ° C. for 20 hours in environmentally controlled conditions.

  Standard ELISPOT tests known to those skilled in the art were used to detect the number of cells producing cytokines IL-4, IL-2, and INF-γ.

Flow cytometric analysis was used to detect INF-γ produced by CD4 + T cells. Methods of using fluorescent color cell sorters (FACS) are well known to those skilled in the art. Briefly, a spleen cell suspension with a concentration of 2.5 × 10 ⁶ cells / ml was prepared and 2 ml per sample was dispensed into individual tubes. Samples stimulated with antigen were then prepared and then incubated at 37 ° C. for 5 hours under environmentally controlled conditions. The sample was then washed and stained prior to reading on a standard FACS reader.

  Standard ELISA tests known to those skilled in the art were used to detect the titer of specific antibodies in sera collected from animals prior to sacrifice.

[Example 1.1: Recombinant hepatitis B surface antigen (HBsAg) adw]
The results in Table 6 below are the results of an ELISPOT test in which the number of cells that produced INF-γ, IL-2, and IL-4 was detected using an HBs antigen adw type recombinant protein. The data in Table 6 (see also FIGS. 1, 2 and 3) represent ELISPOT readings, ie, the number of cells that formed spots, ie, a direct measure of the number of cells that produced cytokines. Yes.

  The number of cells that formed spots was clearly increased by the addition of PIKA adjuvant (compared to antigen alone), indicating that the expression of cytokines INF-γ, IL-2 and IL-4 by cultured spleen cells combined with PIKA adjuvant. It is shown that it was enhanced by adding to the replacement hepatitis B surface antigen. The observed expression of cytokines indicates that the presence of PIKA adjuvant induced a more adaptive immune response in both humoral and cellular immunity.

  The results of an ELISA test on blood samples taken before sacrifice (Table 7 below and FIG. 4) show that the presence of PIKA significantly enhances the immune response and that the specific antibody titer detected in the serum Clarify what was shown.

  The conclusions drawn are that the addition of PIKA adjuvant enhances the overall immune response to the HBs antigen, in particular a specific immune response, more particularly adaptive immunity, and more clearly an immune response significantly biased towards Th1, The cellular immune response is promoted.

[Example 1.2: Inactivated and purified influenza antigen, VAXIGRIP (Sanofi Pasteur), including H1N1, H3N2-like strain and b / Shanghai5 / 361/2002 strain]
The results in Table 8 below show that cells produced INF-γ, IL-2 and IL-4 using the VAXIGIPIP vaccine, an inactivated and split human influenza vaccine manufactured by Sanofi Pasteur. It is the result of the ELISPOT test which detected the number of existence. The data in Table 8 (see also FIGS. 5, 6 and 7) represent ELISPOT readings, ie, the number of cells that formed spots, ie, a direct measure of the number of cells that produced cytokines. Yes.

  The number of cells that formed spots was clearly increased by the addition of PIKA adjuvant (compared to the antigen alone), indicating that the expression of cytokines INF-γ, IL-2 and IL-4 by cultured spleen cells made PIKA adjuvant influenza It is shown that it was enhanced by adding to the antigen. The observed expression of cytokines indicates that the presence of PIKA adjuvant induced a more adaptive immune response in both humoral and cellular immunity.

  The results of an ELISA test on blood samples taken before sacrifice (Table 9 below and FIG. 8) show that the presence of PIKA significantly enhances the immune response and that the specific antibody titer detected in the serum Clarify what was shown.

  The conclusion that can be drawn is that the addition of PIKA adjuvant enhances the overall immune response to influenza antigens, particularly specific immune responses, more particularly adaptive immunity, and more specifically cellular immune responses.

  VAXIGRIP is an approved influenza vaccine that has been found to significantly reduce the risk of infection with influenza. Increased cytokine production with the addition of PIKA indicates that vaccines containing VAXIGIP and PIKA further elicit an immune response that significantly reduces the risk of infection with influenza.

[Example 1.3: Synthetic HIV peptide antigen]
The results in Table 10 below are the results of an ELISPOT test in which the number of cells that produced INF-γ, IL-2, and IL-4 using HIV peptide antigens was detected. The data in Table 10 (see also FIGS. 9, 10 and 11) represent ELISPOT readings, ie, the number of cells that formed spots, ie, a direct measure of the number of cells that produced cytokines. Yes.

  The number of cells that formed spots was clearly increased by the addition of PIKA adjuvant (compared to antigen alone), indicating that the expression of cytokines INF-γ, IL-2 and IL-4 by cultured spleen cells It is clarified that it was enhanced by adding to the HIV antigen. The observed expression of cytokines indicates that the presence of PIKA adjuvant induced a more adaptive immune response in both humoral and cellular immunity.

  The results of FACS analysis are shown in Table 11 below (see also FIG. 12). The presence of CD4 + T cells expressing INF-γ only in formulations containing both PIKA and HIV antigens indicates that the adaptive immune response has reached maturity and that PIKA is beneficial to this process. Make sure your findings.

  The conclusion that can be drawn is that the addition of PIKA adjuvant to the HIV antigen enhances the overall immune response, in particular the specific immune response, more particularly the adaptive immunity, and more specifically the cellular immune response.

[Example 1.4: Recombinant anthrax protective antigen (rPA) from Bacillus anthracis]
The results in Table 12 below are the results of an ELISPOT test in which the presence of INF-γ, IL-2 and IL-4 was detected using recombinant anthrax. The data in Table 12 (see also FIGS. 13, 14 and 15) represent ELISPOT readings, ie, the number of cells that formed spots, ie, a direct measure of the number of cells that produced cytokines. Yes.

  The apparent increase in the number of cells that formed spots (as compared to the antigen alone) by the addition of PIKA adjuvant indicates that the expression of cytokines INF-γ, IL-2 and IL-4 by cultured spleen cells is It is clarified that it was enhanced by adding an adjuvant to the anthrax antigen. The observed expression of cytokines indicates that the presence of PIKA adjuvant induced a more adaptive immune response in both humoral and cellular immunity.

  The results of FACS analysis are shown in Table 13 below (see also FIG. 16). The presence of CD4 + T cells expressing INF-γ only in formulations containing both PIKA and rPA antigens indicates that the adaptive immune response has reached maturity and that PIKA is beneficial to this process Make sure your findings.

  The results of an ELISA test on blood samples taken before sacrifice (Table 14 below and FIGS. 16 and 17) show that the presence of PIKA significantly enhances the immune response and the strength of certain antibodies detected in serum. Clarify what is indicated by the value.

  A standard ELISA test was used to examine the abundance of specific antibodies in blood samples from the original group A and B mice and consistent results were observed 16 weeks after the first vaccine injection. It was. The presence of PIKA along with the anthrax antigen reinduced a significantly higher immune response, as indicated by the titer of specific antibodies in the serum.

  The conclusion that can be drawn is that the addition of PIKA adjuvant enhances the overall immune response to rPA, especially the specific immune response, more particularly the adaptive immunity, and more specifically the cellular immune response.

[Example 1.5: Recombinant herpes simplex virus type 2 gD antigen]
The results in Table 15 below are the results of an ELISPOT test in which the presence of INF-γ, IL-2 and IL-4 was detected using recombinant herpes simplex virus antigens. The data in Table 15 (see also FIGS. 19, 20 and 21) represent ELISPOT readings, ie, the number of cells that formed spots, ie, a direct measure of the number of cells that produced cytokines. Yes.

  The apparent increase in the number of cells that formed spots (as compared to the antigen alone) by the addition of PIKA adjuvant indicates that the expression of cytokines INF-γ, IL-2 and IL-4 by cultured spleen cells is It is clarified that the adjuvant was enhanced by adding the herpes simplex virus antigen. The observed expression of cytokines indicates that the presence of PIKA adjuvant induced a more adaptive immune response in both humoral and cellular immunity.

  The results of FACS analysis are presented in Table 16 below (see also FIG. 22). The presence of CD4 + T cells expressing INF-γ only in formulations containing both PIKA and HSV antigens indicates that the adaptive immune response has reached maturity and that PIKA is beneficial to this process Make sure your findings.

  The results of an ELISA test on blood samples taken before sacrifice (Table 17 below and FIG. 23) show that the presence of PIKA significantly enhances the immune response and that the specific antibody titer detected in the serum Clarify what was shown.

  The conclusion that can be drawn is that the addition of PIKA adjuvant enhances the overall immune response to HSV antigens, in particular specific immune responses, more particularly adaptive immunity, and more specifically cellular immune responses.

[Example 1.6: Whole antigen of inactivated H5N1 virus (avian influenza)]
The results in Table 18 below are the results of an ELISPOT test that detected the presence of INF-γ, IL-2, and IL-4 using inactivated, unpurified H5N1 antigen. The data in Table 18 (see also FIGS. 24, 25 and 26) represent ELISPOT readings, ie, the number of cells that formed spots, ie, a direct measure of the number of cells that produced cytokines.

  The apparent increase in the number of cells that formed spots (as compared to the antigen alone) by the addition of PIKA adjuvant indicates that the expression of cytokines INF-γ, IL-2 and IL-4 by cultured spleen cells is It will be clarified that adjuvant was added to H5N1 antigen. The observed expression of cytokines indicates that the presence of PIKA adjuvant induced a more adaptive immune response in both humoral and cellular immunity.

  The results of FACS analysis are shown in Table 19 below (see also FIG. 27). The presence of INF-γ-expressing CD4 + T cells only in formulations containing both PIKA and H5N1 antigens indicates that the adaptive immune response has reached maturity and that PIKA is beneficial to this process Make sure your findings.

  The results of an ELISA test on blood samples taken before sacrifice (Table 20 below and FIG. 28) show that the presence of PIKA significantly enhances the immune response and that the specific antibody titer detected in the serum Clarify what was shown.

  The conclusion that can be drawn is that the addition of PIKA adjuvant enhances the overall immune response to the H5N1 antigen, in particular specific immune responses, more particularly adaptive immunity, and more specifically cellular immune responses.

[Example 2: SARS antigen of whole inactivated virus]
The purpose of this experiment is to reveal that adding PIKA to SARS antigens enhances the immune response and stimulates the host immune system to produce SARS-specific protective antibodies.

  In this study plan, six groups, each containing 4 Balb / c mice, were subject to SARS antigens, antibodies to PIKA (mainly heterogeneous due to PIKA molecules in the weight distribution range of 66 kDa to 1,200 kDa). (Composition), PIKA alone, or control combinations were inoculated (peritoneal injection) (see also Table 21 below and FIG. 29).

  Each group received the same dose on day 0, day 14 and day 28. At 6 weeks blood samples were extracted and tested for IgG abundance, an indicator of the presence of disease-specific antibodies, in the serum. Serum was diluted by a factor of 16,000 and then IgG abundance was measured using an ELISA reader in a manner well known to those skilled in the art. The output is absorbance (OD), and the larger the value, the greater the amount of IgG present.

  The average results for each group shown in Table 21 reveal the relationship between the presence of PIKA adjuvant and increased IgG expression.

  The conclusion is that the presence of PIKA together with the SARS antigen increases IgG expression in a dose-dependent manner, thereby enhancing the host immune response.

[Example 3: PIKA vaccine provides immune protection against H5N1 and H9N2 infections]
The purpose of this experiment is to demonstrate that avian influenza vaccines containing PIKA adjuvants can protect chickens from infection with live avian influenza viruses.

  The study was conducted on each of the 24 specific pathogen removal (SPF) chickens in the two groups. A dose of 700 ul of vaccine containing PIKA (a heterogeneous composition of PIKA molecules that are mainly in the weight distribution range of 66 kDa to 660 kDa) and two strains of avian influenza (H5N1 and H9N2) The chicken neck was inoculated subcutaneously. The composition contained antigen and PIKA adjuvant in a ratio of about 2: 1 (antigen to PIKA adjuvant).

  On days 7, 14, and 21, blood samples were taken from under the wings. Serum from each chicken was tested for the presence of specific H5 and H9 antibodies.

  On day 21, chickens were inoculated with H5N1 live virus and then observed for an additional 14 days. The survival rate of chickens 14 days after exposure to live H5N1 was recorded.

  From the average results for each group (see also Table 22, FIGS. 30 and 31), it is clear that the presence of PIKA induces the production of antibodies of specific antigens.

  Of the 24 chickens vaccinated with the antigen / PIKA composition, 21 (83%) survived 14 days after exposure to live H5N1 virus. Of the 24 chickens in the control group that received no vaccine but was exposed to live H5N1 virus, only 4 (17%) survived after 14 days.

  The conclusion that is drawn is that the PIKA vaccine provides an effective level of immune protection against H5N1 virus.

[Example 4: PIKA vaccine provides immune protection against rabies infection]
The purpose of this study is to demonstrate that rabies vaccines containing PIKA adjuvants can provide protection against rabies infection.

  From the average results for each group (see also Table 22, FIGS. 30 and 31), it is clear that the presence of PIKA induced the production of antibodies of specific antigens.

  Of the 24 chickens vaccinated with the antigen / PIKA composition, 21 (83%) survived 14 days after exposure to live H5N1 virus. Of the 24 chickens in the control group that received no vaccine but was exposed to live H5N1 virus, only 4 (17%) survived after 14 days.

  The conclusion that is drawn is that the PIKA vaccine provides an effective level of immune protection against H5N1 virus.

[Example 4: PIKA vaccine provides immune protection against rabies infection]
The purpose of this study is to demonstrate that rabies vaccines containing PIKA adjuvants can provide protection against rabies infection.

  Four groups (represented as i, ii, iii, and iv) containing 20 Balb / c SPF Kunming mice were each inoculated with 100 ul of wild rabies virus strain CQ92. Each group is vaccinated with a different type of vaccine; i) PIKA (a heterogeneous composition of PIKA molecules that are primarily in the weight distribution range of 66 kDa to 660 kDa) and inactivated and purified hamster kidney cells A composition comprising a 1: 4 volume ratio of rabies antigen according to ii) inactivated rabies vaccine inactivated by Vero cells, sanofi-aventis Veroab, iii) inactivated and purified comprising alum adjuvant Rabies vaccine with hamster kidney cells, and iv) Control phosphate buffer. A dose of 60 ul of the vaccine was administered 30-40 minutes, 3 days, 6 days and 9 days after infection by subcutaneous injection into the thigh.

  The survival rate of each group is shown in Table 23 (see also FIG. 32).

  The conclusion that can be drawn is that the presence of PIKA significantly enhances the immune defense provided by rabies antigen by inactivated and purified hamster kidney cells.

[Example 5: PIKA hepatitis B vaccine induces production of specific antibodies in serum]
In the protocol of this experiment, three groups of Balb / c mice (three mice per group), group A contains only 4 ug of HBs antigen adw, group B contains 75 ug of PIKA adjuvant (mainly A composition comprising 4 ug of the antigen comprising a heterogeneous composition of PIKA molecules in the weight distribution range of 66 kDa to 1,200 kDa) and Group C subcutaneously injected with a composition comprising only 100 ug of PIKA Vaccinated with

  Mice were given the same booster vaccine by subcutaneous injection 10-14 days after the first injection. Blood samples were taken 10-14 days after the booster injection and tested for specific antibody titers by standard ELISA tests known to those skilled in the art.

  The average results for each group shown in Table 24 below (and FIG. 33) indicate that the immune response to hepatitis B antigen is present in PIKA as indicated by the specific antibody titer observed in the serum samples. It is clarified that it is enhanced by.

  The conclusion drawn from the above example is that immunogenic substances, including PIKA and hepatitis B antigen, induce the production of significant immune responses, as indicated by the titer of specific antibodies in the serum. .

[Example 6: PIKA influenza vaccine induces production of specific antibodies in serum]
In the protocol of this experiment, two groups of Balb / c mice (three mice per group), a composition containing only 4 ug of Sanofi VAXIGIPIP influenza vaccine in group A, and 100 ug of PIKA in group B Vaccinated by subcutaneous injection with a composition containing 4 ug of antigen containing an adjuvant (mainly a heterogeneous composition of PIKA molecules within a weight distribution range of 66 kDa to 1200 kDa).

  Mice received the same booster vaccine by subcutaneous injection 20 days after the first injection. Blood samples were taken 35 days after the first vaccination and tested for specific antibody titers by standard ELISA tests known to those skilled in the art.

  The average results for each group shown in Table 25 (and FIG. 34) below show that the immune response to influenza vaccine antigens is due to the presence of PIKA, as shown by the specific antibody titer observed in the serum samples. It is revealed that it increases.

  The conclusion drawn from the above example is that immunogenic substances, including PIKA and influenza antigens, induce the production of significant immune responses, as shown by the titer of specific antibodies in the serum.

[Example 7: PIKA type B antigen (surface antigen adw type) vaccine induces a therapeutic immune response]
In the protocol of this experiment, groups of 4 Balb / c mice for 6-10 weeks contain PIKA adjuvant (a heterogeneous composition of PIKA molecules that are mainly in the weight distribution range of 66 kDa to 1200 kDa). It is necessary to inoculate a composition containing a commercially available HBs antigen adw type, a composition containing an antigen without PIKA adjuvant, a composition containing only PIKA, or a control containing phosphate buffer.

  Mice were given a first subcutaneous injection of 100 ul on each side of the back. The actual dosage is given in the results table below. Mice were given the same booster vaccine 21 days after the first injection. Blood samples were collected on day 42, after which the mice were sacrificed and tissue samples were taken from the spleen for examination.

  ELISPOT analysis was performed to count the number of T cells secreting antigen-specific interferon-γ. In order to determine the presence of peptide epitopes derived from HBs antigen (IPQSLDSWWTSL) specific CD8 + cells, splenocyte samples from each mouse were obtained at a concentration of 5 ug / ml of either CD8 T cell peptide epitope (IPQSLDSWWTSL). ) To stimulate in vitro.

  The second splenocyte sample was re-stimulated with 2 ug / ml HBs antigen peptide IPQSLDSWWTSL for 6 days in vitro. To detect interferon-γ, ELISPOT analysis was performed using 5 ug / ml of HBs antigen peptide IPQSLDSWWTSL as an in vitro stimulus. This analysis was performed to evaluate and identify the central memory cell response following immunization.

  ELISA analysis was used to determine the presence of HBV antigen specific antibodies in serum, particularly IgG1 and IgG2a antibodies. Nunc Immunoplate Maxisorp plates were coated overnight at 4 degrees Celsius with HBs antigen (6 ug / ml in PBS / 0.01% Tween 20). Plates were washed with PBS / Tween and blocked for 2 hours with PBS containing 5% FCS. After washing, a diluted solution of serum with PBS / Tween was added for 2 hours. After washing, either a 1/3000 dilution of a rat anti-mouse IgG1 monoclonal antibody conjugated with biotin or a 1/1500 dilution of a rat anti-mouse IgG2a monoclonal antibody conjugated with biotin was added. After washing, streptavidin HRP (1 / 10,000 dilution with PBS / Tween) was added for 1 hour. After washing, ABTS substrate was added with hydrogen peroxide (1000: 1) for 20 minutes. The absorbance (OD) at 405 nm was then measured and the results shown are the average in each group.

  The IgG1 response in mice immunized with HBs antigen formulated with PIKA adjuvant was approximately 5-fold higher than in mice immunized with HBs antigen alone. IgG1 titers increased in a dose-dependent manner (Table 26, FIG. 35).

  The IgG2a response in mice immunized with HBs antigen formulated with PIKA adjuvant was significantly increased over that in mice immunized with HBs antigen alone. The titer of IgG2a increased in a dose-dependent manner, indicating that the Th1-biased immune response increased (Table 27, FIG. 36).

  ELISPOT analysis on CD8 peptide epitope-specific ex vivo stimulation showed that no response was detected by mice immunized with HBs antigen alone. In contrast, after immunization with HBs antigen formulated with PIKA, the cells that express interferon-γ were easily detected in a dose-dependent manner, and the therapeutic immune response is enhanced by PIKA. (Table 28, FIG. 37).

  The number of splenocytes producing interferon-γ in mice immunized with a preparation containing HBs antigen and PIKA adjuvant was determined by ELISPOT analysis further performed after culturing splenocytes for 6 days. It was revealed that it was twice the number of splenocytes in. The above results confirm that the presence of PIKA enhances the activation of central memory cells (Table 28, FIG. 38).

FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with a vaccine containing PIKA and / or HBs antigen adw type. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes producing IL-2 after immunization with a vaccine containing PIKA and / or HBs antigen adw type. FIG. 4 is an ELISPOT detection of IL-4 produced by mouse splenocytes after immunization with a vaccine containing PIKA and / or HBs antigen adw type. FIG. 5 is a diagram of ELISPOT detection of specific IgG titers derived from mouse serum (400 × dilution) after immunization with vaccines containing PIKA and / or HBs antigen adw type. FIG. 7 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-gamma (γ) after immunization with a vaccine containing PIKA and / or inactivated and split influenza antigen. FIG. 10 is an ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with a vaccine containing PIKA and / or inactivated and split influenza antigen. FIG. 5 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with a vaccine containing PIKA and / or inactivated and split influenza antigen. FIG. 10 is an ELISA detection of specific IgG titers derived from mouse serum (900 × dilution) after immunization with vaccines containing PIKA and / or inactivated and split influenza antigen. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with a vaccine containing PIKA and / or HIV gp120 antigen. FIG. 5 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with a vaccine containing PIKA and / or HIV gp120 antigen. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with a vaccine containing PIKA and / or HIV gp120 antigen. FIG. 5 is a diagram of FACS analysis in mouse splenocytes after immunization with a vaccine containing PIKA and / or HIV gp120 antigen, showing the percentage of CD4 + cells expressing interferon-γ. FIG. 5 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with a vaccine containing PIKA and / or anthrax rPA antigen. FIG. 6 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with a vaccine containing PIKA and / or anthrax rPA antigen. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with a vaccine containing PIKA and / or anthrax rPA antigen. FIG. 5 is a diagram of FACS analysis in mouse splenocytes after immunization with a vaccine containing PIKA and / or anthrax rPA antigen, showing the percentage of CD4 + cells expressing interferon-γ. FIG. 6 is an ELISA detection of specific IgG titers derived from mouse serum (400 × dilution) after immunization with a vaccine containing PIKA and / or anthrax rPA antigen. FIG. 6 is an ELISA detection of specific IgG titers from mouse serum 16 weeks after immunization with vaccines containing PIKA and / or anthrax rPA antigen. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with a vaccine containing PIKA and / or HSV2-gD antigen. FIG. 5 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with a vaccine containing PIKA and / or HSV2-gD antigen. FIG. 4 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with a vaccine containing PIKA and / or HSV2-gD antigen. FIG. 6 is a diagram of FACS analysis in mouse splenocytes after immunization with a vaccine containing PIKA and / or HSV 2gD antigen, showing the percentage of CD4 + cells expressing interferon-γ. FIG. 5 is an ELISA detection of specific IgG titers derived from mouse serum (2,700 × dilution) after immunization with a vaccine containing PIKA and / or HSV2-gD antigen. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with a vaccine containing PIKA and / or inactivated H5N1 antigen. FIG. 2 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-2 after immunization with a vaccine containing PIKA and / or inactivated H5N1 antigen. FIG. 4 is a diagram of ELISPOT detection of mouse splenocytes that produced IL-4 after immunization with a vaccine containing PIKA and / or inactivated H5N1 antigen. FIG. 5 is a diagram of FACS analysis in mouse splenocytes after immunization with a vaccine containing PIKA and / or inactivated H5N1 antigen, showing the percentage of CD4 + ve cells expressing interferon-γ. FIG. 5 is an ELISA detection of specific IgG titers from mouse serum (900 × dilution) after immunization with vaccines containing PIKA and / or inactivated H5N1 antigen. FIG. 5 is an ELISA detection of specific IgG titers derived from mouse serum (16,000 dilution) after immunization with a vaccine containing whole PIKA and / or inactivated SARS antigen. FIG. 6 is an ELISA detection of the titer of specific antibody H5 derived from chicken serum after immunization with a vaccine containing PIKA and / or inactivated H5N1 antigen. FIG. 9 is an ELISA detection of the titer of specific H9 antibodies derived from chicken serum after immunization with a vaccine containing PIKA and / or inactivated H9N2 antigen. FIG. 6 is a survival rate diagram of mice prescription for rabies vaccine after exposure to wild rabies virus. FIG. 5 is an ELISA detection of the titer of specific antibodies derived from mouse serum after immunization with a vaccine containing PIKA and / or HBs antigen adw. FIG. 5 is an ELISA detection of specific antibody titers from mouse sera after immunization with a vaccine containing PIKA and / or inactivated and split influenza antigen. FIG. 6 is an ELISA detection of specific IgG1 titers from mouse serum after immunization with vaccines containing PIKA and / or HBs antigens. FIG. 6 is an ELISA detection of specific IgG2 titers from mouse serum after immunization with vaccines containing PIKA and / or HBs antigens. FIG. 4 is a diagram of ELISPOT detection of mouse splenocytes that produced interferon-γ after immunization with a vaccine containing PIKA and / or HBs antigen. FIG. 6 is a diagram of ELISPOT detection of mouse splenocytes (after 6 days restimulation with 2 ug / ml IPQ peptide) that produced interferon-γ after immunization with vaccines containing PIKA and / or HBs antigen.

Claims (23)

(A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) an immunogenic composition comprising at least one antigen,
An immunogenic composition characterized by being formulated for sustained release. (A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) an immunogenic composition comprising at least one viral antigen,
The above antigens are adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, flaviviridae, hepatitis viridae, hepeviridae, mononegaviridae, nidoviruses, picornaviridae, ortho An immunogenic composition characterized by being an antigen of Myxoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, or Togaviridae. (A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) at least one bacterial antigen;
An immunogenic composition, wherein the antigen is an actinomycete, Chlamydia, Gram-positive bacterium, proteobacteria, or spirocator. (A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) including at least one fungal antigen;
An immunogenic composition, wherein the antigen is an ascomycota or basidiomycete antigen. (A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) at least one parasite antigen,
An immunogenic composition, characterized in that the antigen is a fleshly flagellate, apicomplexa, ciliate, flatworm, linear or arthropoda antigen. (A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) including at least one cancer antigen;
The antigen is osteosarcoma, brain tumor, breast cancer, gastrointestinal / gastrointestinal cancer, endocrine adenocarcinoma, eye cancer, genitourinary cancer, germ cell tumor, gynecological cancer, head and neck cancer, fluid / hematoma An immunogenic composition characterized by being a cancer antigen of lung cancer, musculoskeletal tumor, neuronal cancer, respiratory / chest cancer or skin cancer. (A) a polynucleotide adjuvant comprising polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic and at least one cation;
(B) including a combination of two or more antigens,
The above antigens are adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, flaviviridae, hepatitis viridae, hepeviridae, mononegaviridae, nidoviruses, picornaviridae, ortho Myxoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Togaviridae, Actinomyces, Chlamydia, Gram-positive bacteria, Proteobacteria, or Spirocator An immunogenic composition characterized in that it is an antigen of a phylum, Ascomycota or Basidiomycota, fleshly flagellate, apicomplexa, trichomes, flatworm, linear phylum and / or arthropoda .   8. The immunogenic composition of any one of claims 1 to 7, comprising a polynucleotide adjuvant composition molecule of heterogeneous molecular weight, wherein the molecular weight is at least 66,000 daltons.   9. The polynucleotide adjuvant composition molecule of heterogeneous molecular weight, wherein the molecular weight is from about 66,000 to 1,200,000 daltons. An immunogenic composition.   10. The immunogenic composition according to any one of claims 1 to 9, comprising a polynucleotide adjuvant composition molecule of heterogeneous molecular weight, wherein the molecular weight is at least 150,000 daltons.   The immunogenic composition according to any one of claims 1 to 10, further comprising at least one immunostimulant.   The immunogenic composition according to any one of claims 1 to 11, wherein the antigen is an inactivated microorganism, an attenuated microorganism, a recombinant polypeptide and / or a synthetic polypeptide.   The immunogenic composition according to any one of claims 1 to 12, wherein the immunogenic composition or the PIKA adjuvant contained in the immunogenic composition is a liquid, solution, droplet, solid Product, capsule, emulsion, suspension, elixir, cream, suppository, gel, soft capsule, spray, inhalation antigen, aerosol, tablet, coated tablet, microcapsule, suppository, sugar-coated tablet, syrup, slurry, enema, granule, An immunogenic composition characterized in that it is in the form of a powder, tablet or troche.   The immunogenic composition according to any one of claims 1 to 12, wherein at least one of the adjuvant compositions or the immunogenic composition is lyophilized. Sex composition.   A kit comprising the immunogenic composition according to any one of claims 1 to 14. A method for enhancing a host immune response to an antigen, comprising:
15. A method comprising administering an immunogenic composition according to any one of claims 1 to 14 to a host.   The method according to claim 16, wherein the host is suffering from an infectious disease, and administration of the antigenic compound causes an immune response against the pathogen causing the infectious disease.   The method according to claim 16 or 17, wherein the administration is by parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, topical delivery, transdermal delivery or intradermal delivery.   Use of the immunogenic composition according to any one of claims 1 to 14 for preparing a medicament for enhancing an immune response in a host to the antigen.   The use according to claim 19, wherein the host suffers from an infectious disease, and administration of the antigen to the host causes an immune response against the pathogen causing the infectious disease.   Use according to claim 20, characterized in that the administration is by parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, topical delivery, transdermal delivery or intradermal delivery.   The method according to any one of claims 16 to 18 and the use according to any one of claims 19 to 21, wherein the host is a human.   The method according to any one of claims 16 to 18 and the use according to any one of claims 19 to 21, wherein the host is a non-human animal.

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